Pharmacophore recombination for the identification of small molecule drug lead compounds

ABSTRACT

A library of candidate target binding fragments (CTBF&#39;s) each CTBF being a small organic molecule and having a linkable functional group (LFG) or blocked form thereof (BLFG), wherein the LFG or BLFG contains a linking group (LG), the LG being a disulfide group.

[0001] This invention was made with Government support under ContractNo. R01 GM50353 awarded by the National Institutes of Health. TheGovernment has certain rights to this invention.

FIELD OF THE INVENTION

[0002] The present invention is directed to novel methods foridentifying small molecule drug lead compounds.

BACKGROUND OF THE INVENTION

[0003] In response to the ever increasing demand for novel compoundsuseful in the effective treatment of various maladies, the medicalresearch community has developed a number of different strategies fordiscovering and optimizing new therapeutic drugs. For the most part,these strategies are dependent upon molecular techniques that allow theidentification of tightly binding ligands for a given biological targetmolecule. Once identified, these ligands may then carry out theirtherapeutic functions by activating, inhibiting or otherwise alteringthe activity of the molecular target to which they bind.

[0004] In one such strategy, new therapeutic drugs are identified byscreening combinatorial libraries of synthetic small molecule compounds,determining which compound(s) have the highest probability of providingan effective therapeutic and then optimizing the therapeutic propertiesof the identified small molecule compound(s) by synthesizingstructurally related analogs and analyzing them for binding to thetarget molecule (Gallop et al., J. Med. Chem. 37:1233-¹²⁵I (1994),Gordon et al., J. Med. Chem. 37:1385-1401 (1994), Czarnik and Ellman,Acc. Chem. Res. 29:112-170 (1996), Thompson and Ellman, Chem. Rev.96:555-600 (1996) and Balkenhohl et al., Angew. Chem. Int. Ed.35:2288-2337 (1996)). However, this process is not only time consumingand costly, it often does not provide for the successful identificationof a small molecule compound having sufficient therapeutic potency forthe desired application. For example, while the preparation andevaluation of combinatorial libraries of small molecules has provensomewhat useful for new drug discovery, the identification of smallmolecules for difficult molecular targets (e.g., such as those usefulfor blocking or otherwise taking part in protein-protein interactions)has not been particularly effective (Brown, Molecular Diversity2:217-222 (1996)).

[0005] One issue that limits the success of combinatorial libraryapproaches is that it is possible to synthesize only a very smallfraction of the possible number of small molecules. For example, greaterthan 10⁶⁰ different small molecules having valid chemical structures andmolecular weights under 600 daltons can be envisioned. However, even themost ambitious of small molecule combinatorial library efforts have beenable to generate libraries of only tens to hundreds of millions ofdifferent compounds for testing. Therefore, combinatorial technologyallows one to test only a very small subset of the possible smallmolecules, thereby resulting in a high probability that the most potentsmall molecule compounds will be missed. Thus, suitable small moleculecompounds having the required availability, activity or chemical and/orstructural properties often cannot be found. Moreover, even when suchsmall molecule compounds are available, optimization of those compoundsto identify an effective therapeutic often requires the synthesis of anextremely large number of structural analogs and/or prior knowledge ofthe structure of the molecular target for that compound. Furthermore,screening large combinatorial libraries of potential binding compoundsto identify a lead compound for optimization can be difficult andtime-consuming because each and every member of the library must betested. It is evident, therefore, that novel methods for rapidly andefficiently identifying new small molecule drug leads are needed.

[0006] Living organisms evolve through a process that includes both (1)genetic recombination, where sexual reproduction acts to mix andrecombine the attributes of the parent organisms to provide progenyhaving attributes of both parents, and (2) natural selection, where onlythose progeny that are sufficiently “fit” are capable of passing theirattributes on to the next generation. Approaches that closely model theprocess by which organism evolve have previously been reported foridentifying small molecules that bind to receptors and enzymes (Weber etal., Angew. Chem. Int. Ed. Engl. 34:2280-2282 (1995) and Singh et al.,J. Am. Chem Soc. 118:1669-1676 (1996)). These approaches are based uponthe mathematical method termed “genetic algorithms” (Holland, Sci. Am.66-72 (1992)). Using genetic algorithms, a population of differentcompounds is screened to identify the compounds that bind to thereceptor or enzyme (i.e., the “fittest” compounds). A population ofprogeny compounds is then prepared by recombining the building blocksthat were used to prepare the “fittest” compounds. A screen is thenperformed to identify the compounds that bind to the target with thehighest affinity, which are made up of the optimal building blockcombinations.

[0007] However, because the building blocks employed in the geneticalgorithm approach are not preselected, one of two techniques are usedto identify tight binding ligands: (1) extremely large populations ofcompounds must be screened and recombined, or (2) multiple rounds ofscreening and recombination are performed on relatively smallpopulations where additional building blocks are gradually introducedthrough a process that is analogous to genetic mutation. In this secondapproach, many rounds of selection, recombination and building blockintroduction are required to identify the optimal building blockcombinations in analogy to the many rounds of selection, reproductionand mutation that are required in the evolution of living organisms.Thus, the use of genetic algorithms is currently limited because of thelarge amount of time required for compound preparation and screening,wherein the goal of new drug discovery is to identify a potent compoundas quickly as possible.

[0008] Another recently reported approach for identifying high affinityligands for molecular targets of interest is by determiningstructure-activity relationships from nuclear magnetic resonanceanalysis, i.e., “SAR by NMR” (Shuker et al., Science 274:1531-1534(1996) and U.S. Pat. No. 5,698,401 by Fesik et al.). In this approach,the physical structure of a target protein is determined by NMR and thensmall molecule building blocks are identified that bind to the proteinat nearby points on the protein surface. Adjacently binding smallmolecules are then coupled together with a linker in order to obtaincompounds that bind to the target protein with higher affinity than theunlinked compounds alone. Thus, by having available the NMR structure ofthe target protein, the lengths of linkers for coupling two adjacentlybinding small molecules can be determined and small molecule ligands canbe rationally designed. This approach has been useful for identifyingcompounds that bind to FK506 binding protein with a K_(d)=20 nM (Shukeret al., supra) and to stromelysin with a K_(d)=15 nM (Hajduk et al., J.Am. Chem. Soc. 119:5818-5827 (1997) and Hajduk et al., J. Am. Chem. Soc.119:5828-5832 (1997)).

[0009] However, while the SAR by NMR method is powerful, it also hasserious limitations. For example, the approach requires huge amounts oftarget protein (>200 mg) and this protein typically must be ¹⁵N-labeledso that it is useful for NMR studies. Moreover, the SAR by NMR approachusually requires that the target protein be soluble to>0.3 mM and have amolecular weight less than about 25-30 K_(d)a. Additionally, thestructure of the target protein is first resolved by NMR, a processwhich often can require a 6 to 12 month time commitment.

[0010] From the above, it is evident that there is a need for noveltechniques useful for rapidly and efficiently identifying small moleculedrug lead compounds that are capable of binding with high affinity to amolecular target of interest. We herein describe for the first time amethod which is based upon pharmacophore recombination, wherein apopulation of small molecule pharmacophores are “preselected” for theability to bind to a molecular target and wherein the small moleculepharmacophores that bind with the highest affinity are then chemicallylinked in various combinations to provide a library of potential highaffinity binding ligands. The library of potential binding ligands isthen screened using a simple functional assay for the presence of one ormore compounds that bind to the target molecule with very high affinity.

SUMMARY OF THE INVENTION

[0011] Applicants herein describe a molecular approach for rapidly andefficiently identifying small molecule ligands that are capable ofbinding to a target biological molecule with high affinity, whereinligand compounds identified by the method are useful as new smallmolecule drug lead compounds. The herein described methods allow alibrary of only the most favorable compounds to be assayed for bindingto a target biological molecule without the need for screening allpossible small molecule compounds and combinations thereof for bindingto the target as is required in standard combinatorial libraryapproaches. More specifically, a library of candidate target bindingfragments is assembled and subjected to a first screen or “pre-screened”to identify a subset of that library that bind to a target biologicalmolecule with or below a certain dissociation constant. Those candidatetarget binding fragments identified during this “pre-screening” step asbeing capable of binding to the target biological molecule are thencoupled or cross-linked in a variety of combinations using one or morelinker elements to provide a library of potential high affinity bindingligands or candidate cross-linked target binding fragments, whosebuilding blocks represent the small candidate target binding fragmentshaving the highest affinity for the target biological molecule asidentified in the “pre-screening” step. The library of potential ligandsor candidate cross-linked target binding fragments for binding to thetarget biological molecule is then screened a second time to identifythose members that exhibit the lowest dissociation constant for bindingto the target biological molecule. Because the library of candidatetarget binding fragment building blocks is initially “pre-screened” toselect for a much smaller set of the most favorable building blocks, themost productive building block and cross-linker combinations can beidentified without the laborious task of screening all possiblecombinations of all building blocks coupled together by a set oflinkers. The process of identifying high affinity drug lead compounds istherefore, greatly expedited.

[0012] With regard to the above, one embodiment of the present inventionis directed to a method for identifying drug lead compounds that bind toa biological target molecule of interest, wherein the method comprisesthe steps of:

[0013] (a) Assembling a library of candidate target binding fragments(CTBF) capable of being chemically cross-linked by a cross-linkerelement to provide candidate cross-linked target binding fragments forbinding to the target biological molecule;

[0014] (b) screening the library of candidate target binding fragmentsto identify at least first and second candidate target binding fragmentswhich bind to the target biological molecule;

[0015] (c) chemically cross-linking the at least first and secondcandidate target binding fragments or structurally related analogsthereof with a cross-linker element to provide a library of candidatecross-linked target binding fragments for binding to the targetbiological molecule; and

[0016] (d) screening the library obtained in (c) to identify a drug leadcompound that binds to the target biological molecule.

[0017] In various preferred embodiments, the library of candidate targetbinding fragments may comprise compounds of less than 500 daltons, maycomprise simple aldehydes, amines, amides, carbamates, ureidos,sulfonamides, alcohols, carboxylic acids, thiols, aryl halides, alkenes,alkynes, ketones, ethers and/or oximes and/or may bind to the targetbiological molecule with a K_(d) of 10 mM or lower. In a particularlypreferred embodiment, the library or candidate target binding fragmentsmay comprise oxime compounds, wherein the structurally related aldehydeanalogs of those oxime compounds are capable of being chemicallycross-linked via an O,O′-diamino-alkanediol cross-linker. Targetbiological molecules that find use in the described methods include, forexample, proteins, nucleic acids and saccharides, preferably proteins.Preferred TBM's include human or human pathogen proteins, especiallyenzymes, human hormones, human receptors and fragments thereof. TheseTBM's may all contain atoms of naturally occuring isotopic abundance.

[0018] In other preferred embodiments, the library of candidatecross-linked target binding fragments comprises candidate cross-linkedtarget binding fragments of less than about 1000 daltons, that may behomo- or heterodimeric having a K_(d) for the TBM of from about 500 μMto about 500 nM or lower.

[0019] Another embodiment of the present invention is directed to amethod for inhibiting the interaction between first and secondbiological molecules, wherein the method comprises the step ofcontacting a system comprising both the first and second biologicalmolecules with a binding inhibitory amount of a candidate cross-linkedtarget binding fragment identified by the above described method,wherein the candidate cross-linked target binding fragment binds to oneof the first or second biological molecules and inhibits their abilityto interact.

[0020] A further embodiment of the present invention is directed to adrug lead compound made by the the method described herein, where thecompound is represented by the formula:

[0021] where

[0022] TBF_(m) represents a first TBF selected from step (d);

[0023] TBF_(n) represents a second TBF selected from step (d);

[0024] TBF_(m)-part A and B represent TBF from step (d) where eachfragment is bonded to a single atom in LG₃;

[0025] TBF_(n)-part C and D represent TBF from step (d) where eachfragment is bonded to a single atom in LG₄;

[0026] XL represents a cross-linker of the formula

-(C₀-C₂-alkyl-L¹-L²-L³-L⁴-L⁵-C₁-C₂-alkyl)-;

[0027] LG₁ and LG₂ are linking groups independently selected from thegroup —C(R_(a))═N—O—, —O—N═C(R_(a))—, —CH₂—N(R_(a))—, —N(R_(a))—CH₂—,—C(═O)—N(R_(a))—, —N(R×a)—C(═O)—, N(R_(a))—C(═O)—O—, —O—C(═O)—N(R_(a))—,—N(R_(a))—C(═O)—N(R_(b))—N(R_(a))—C(═O)—N(R_(b))—, SO₂—N(R_(a))— and—N(R_(a))—SO₂—;

[0028] LG₃ and LG₄ are linking groups independently selected from thegroup>C═NO—, —O—N═C<, —CH—N<, >N—CH—, —C(═O)—N<, >N—C(═O)—, >N—C(═O)—O—,—O—C(═O)<, >N—C(═O)—N(R_(b))—, —N(R_(b))—C(═O)—N<, —SO₂—N<and>N—SO₂—,where < and > represent two bonds linking CTBF— part A, B, C, or D tothe single N or C atom in G₃ or LG₄;

[0029] R_(a) and R_(b) are independently selected from the grouphydrogen, C, —C₁₀-alkyl, C₀-C₁₀-alkyl-C₆-C₁₀-aryl,C₆-C₁₀-aryl-C₀-C₁₀-alkyl, C₀-C₁₀-alkyleterocycle-C₀-C₁₀-alkyl,C₁-C₆-alkyl-NH—C₁-C₆-alkyl, C₀-C₁₀-alkyl-O—C₀-C₁₀-alkyl,C₀-C₁₀-alkyl-C(═O)—C₀-C₁₀-alkyl, C₀-C₁₀-alkyl-NH—C(═O)—C₀-C₁₀-alkyl,C₀-C₁₀-alkyl-O-C(═O)-C₀-C₁₀-alkyl, where any alkyl, aryl or heterocycleis optionally substituted with C₁-C₁-alkyl, C₆-C₁₀-alkoxy, C₆-C₁₀-aryl,C₆-C₁₀-aryloxy, halo (F, Cl, Br, I), hydroxy, carboxy, amino, nitro andS(O)₃;

[0030] TBF_(m), TBF_(n), TBF_(m)-part A, TBF_(m)-part B, TBF_(n)-part Cand TBF_(n)-part D are each independently represented by formula I

-A-(Cycle 1)-B-(Cycle 2)-E  (I)

[0031] Where

[0032] Cycle 1 and Cycle 2 are independently present or absent and areselected from a mono-, bi-, or tricyclic saturated, unsaturated, oraromatic ring, each ring having 5, 6 or 7 atoms in the ring where thering atoms are carbon or from 1-4 heteroatoms selected from; nitrogen,oxygen, and sulfur, and where any sulfur ring atom may optionally beoxidized and any carbon ring atom may form a double bond with O, NR^(n)and CR¹R¹, each ring nitrogen may be substituted with R and any ringcarbon may be substituted with R^(d);

[0033] A and B are independently selected from

[0034] -L³-L²-L¹ and

[0035] -L⁴-L³-L²-L¹ and

[0036] -L⁵-L⁴-L³-L²-L¹-.

[0037] where:

[0038] L¹ is absent or may be selected from oxo (O), S(O), C(═O),C(═N—R^(n)), C(═CR¹R¹), C(R¹R^(1′)), C(R¹), C, het, N(R^(n)) or N;

[0039] L³ is absent or may be selected from oxo (O), S(O), C(═O),C(═N—R^(n)), C(═CR²R²¹), C(R²R^(2′)), C(R²), C, het, N(R^(n)) or N;

[0040] L³ is absent or may be selected from oxo (O), S(O)S, C(═O),C(═N—R^(n)) C(═CR³R^(3′)), C(R³R^(3′)) C(R³), C, het, N(R^(n)) or N;

[0041] L⁴ is absent or may be selected from oxo (O), S(O), C(═O),C(═N—R^(n)), C(═CR⁴R^(4′)), C(R⁴R^(4′)), C(R 4), C, NR^(n) or N; and

[0042] L⁵ is absent or may be selected from oxo (O), S(O), C(═O),C(═N—R^(n)), C(R⁵R^(5′)), C(═CR⁵R^(5′)), C(R⁵), C, NR^(n) or N;

[0043] R¹, R^(1′), R², R^(2′), R³, R^(3′), R⁴, R^(4′), R⁵ and R^(5′)each are independently selected from R^(a), R^(a′), R^(c) and U-Q-V-W;where s is 0-2

[0044] Optionally, each R¹—R⁵ or NR^(n) together with any other R¹—R⁵ orNR^(n) may form a mono-, bi-, or tricyclic saturated, unsaturated, oraromatic ring, each ring being a homo- or heterocycle having 5, 6 or 7atoms in the ring, optionally each ring containing 14 heteroatomsselected from N, O and S where any ring carbon or sulfur atom mayoptionally be oxidized, each ring nitrogen optionally substituted withR^(n) and each ring carbon optionally substituted with R^(d);

[0045] E is -L¹-L²-L³-R^(a);

[0046] R^(a) is selected from the group; hydrogen, halo(F, Cl, Br, I),halo(F, Cl, Br, I)-C₁-C₁₁alkyl, halo(F, Cl, Br, I)—C₁-C₁₁alkoxy,hydroxy-C₁-C₁₁alkyl, cyano, isocyanate, carboxy-C₀-C₁₁ alkyl, amino,C₀-C₁₁-alkyl-amino-(C₁-C₈alkyl), C₀-C₁₁alkyl-amino-di-(C₁-C₈alkyl),aminocarbonyl, C₁-C₁₁alkylcarbonylamino, carboxamido, carbamoyl,carbamoyloxy, formyl, formyloxy, azido, nitro, hydrazide, hydroxamicacid, imidazoyl, ureido, thioureido, thiocyanato, hydroxy, C₁-C₆alkoxy,mercapto, sulfonamido, het, phenoxy, phenyl, benzyl, benzyloxy,benzamido, tosyl, morpholino, morpholinyl, piperazinyl, piperidinyl,pyrrolinyl, imidazolyl and indolyl;

[0047] R^(a′) is selected from the group of C₀-C₁₀alkyl-Q-C₀-C₆alkyl,C₀-C₁₀alkenylQ-C₀-C₆ alkyl, C₀-C₁₀alkynyl-Q-C₀-C₆alkyl,C₃-C₁₁cycloalkyl-Q-C₀-C₆alkyl, C₃-C₁₀cycloalkeny-Q-C₀-C₆alkyl,C₁-C₆alkyl-C₆-C₁₂aryl-Q-C₁-C₆alkyl, C₆-C₁ arylC₁-C₆alkyl-Q-C₀-C₆alkyl,C₀-C₆alkyl-het-Q-C₀-C₆alkyl, C₀-C₆alkyl-Q-het-C₀-C₆alkyl,het-C₀-C₆alkyl-Q-C₀-C₆alkyl, C₀-C₁₆ alkyl-Q-C₆-C₁₂aryl and Q-C C₆alky,where any aryl or het is optionally substituted with 1-3 R^(d) and anyalkyl, alkenyl or alkynyl is optionally substituted with 1-3 R^(a);

[0048] R^(a) and R^(a′) may join to form a 3-7 member homocyclic ringsubstituted with 1-3 R^(a);

[0049] R^(c) is selected from hydrogen and substituted or unsubstituted;amino, O-C₁C₈alkyl, amino-(C₁-C₈alkyl), amino-di-(C₁-C₈alkyl),C₁-C₁₀alkyl, C₂-C₁₀alkenyl, C₂-C₁₀alkynyl, C₃-c₁₁cycloalkyl,C₃-C₁₀cycloalkenyl, C₁-C₆alkyl-C₆-C₂aryl, C₆-C₁₀aryl-C₁-C₆alkyl,C₁-C₆alkyl-het, het-C₁-C₆alkyl, C₆-C₁₂aryl and het, where thesubstituits on any alkyl, alkenyl or alkynyl are 1-3 R^(a) and thesubstituents on any aryl or het are 1-3 R^(d);

[0050] R^(d) is selected from R^(h) and R^(p);

[0051] R^(h) is selected from the group OH, OCF₃, OR^(c), SR^(m),halo(F, Cl. Br, I), CN, isocyanate, NO₂, CF₃, C₀-C₆alkyl R^(n′),C₀-C₆alkyl-C(═O)—NR^(n)R^(n′), C₀-C₆alkyl, C₁-C₈alkoxy, C₀C₆-alkenyl,C₀-C₆alkynyl, C₀-C₆alkyl-C(═O)-R^(n′), C₀C₆cycloalkyl,C₃-C₆cycloalkenyl, C₁-C₆alkyl-phenyl, phenyl-C₁-C₆alkyl,C₁C₆alkyloxycarbonylamino, C₁-C₆alkyloxycarbonyl-C₀-C₆alkyl,phenyl-C₀C₆alkyloxy, C₁-C₆alkyl-het, het-C₁-C₆alkyl, SO₂-het,O—C₆-C₁₂aryl, SO₂-C₆-C₁₂ aryl, SO₂—C₀-C₆alkyl and het, where any alkyl,alkenyl or alkynyl may optionally be sustituted with 1-3 groups selectedfrom OH, halo(F, Cl, Br, I), nitro, amino and aminocarbonyl, where thesubstituents on any aryl or het are 1-2 hydroxy, halo(F, Cl, Br, I),CF₃, C₁-C₆alkyl, C₁-C₆alkoxy, nitro and amino;

[0052] R^(m) is selected from hydrogen, S—C₁-C₆alkyl, C(═O)—C₁-C₆alkyl,C(═O)—NR^(n)R^(n′), C₁-C₆alkyl, halo(F, Cl, Br, I)-C₁-C₆alkyl, benzyland phenyl;

[0053] R^(n) is selected from the group R^(c), OH, OCF₃, OR⁰, CN,isocyanate, NH—C(═O)—O—R^(c), NH—C(═O)—R^(c), NH—C(═O)—NHR^(c),NH—SO₂—R^(s), NH—SO₂—NH—C(═O)—R^(c), NH—C(═O)—NH—SO₂—R^(c),C(═O)—O—R^(s), C(═O)—R^(c), C(═O)—NHR^(c), C(═O)—NH₅ C(═O)—O—R^(s),C(═O)—NH—C(═O)—R^(c), C(═O)—NH—SO₂—R⁵, C(═O)—NH—SO₂—NHR^(c), SO₂—R^(s),SO₂—O—R^(O), SO₂—N(R^(c))₂, SO₂—NH—C(═O)—O—R⁰, SO₂—NH—C(═O)—O—R^(O) andSO₂—NH—C(═O)—R^(c);

[0054] R^(O) is selected from hydrogen and substituted or unsubstitutedC₁-C₆alkyl, C₀-C₆alkyl-C₆-C₁₀aryl, C₁-C₆alkylcarbonyl, C₂-C₆ alkenyl,C₂-C₆alkynyl, C₃C₈cycloalkyl and benzoyl, where the substituits on anyalkyl are 1-3 R^(a) and the substituents on any aryl are 1-3 R^(p);

[0055] R^(p) is selected from the group; OH, COOH, COH, NH₂, C₀-C₆alkyl,halo(F, Cl. Br, I), CN, isocyanate, OR^(O), SR^(m), SOR^(O), NO₂, CF₃,R^(c), NR^(n)R^(n′), N(R^(n))_C(═O)—O—R^(O), N(R^(n))—C(═O)—R^(c), SO₂—RC₀-C₆alkyl-SO₂-R^(s), C₀-C₆alkyl-SO 2-NR^(n)R^(n′), C(═O)—R^(c),O—C(═O)—R^(c), C(═O)—O—R^(O) and C(═O)—NR^(n)R^(n′), where thesubstituits on any alkyl, alkenyl or alkynyl are 1-3 R and thesubstituents on any aryl or het are 1-3 R^(d);

[0056] R^(s) is a substituted or unsubstituted group selected from;C₁-C₈alkyl, C₂-C₈alkenyl, C₂-C₈alkynyl, C₃-C₈cycloalkyl,C₃-C₆cycloalkenyl, C₀-C₆alkyl-C₆-C₁₀aryl, C₆-C₁₀aryl-C₀-C₆alkyl,C₀-C₆alkyl-het and het-C₀-C₆alkyl, where the substituits on any alkyl,alkenyl or alkynyl are 1-3 R^(a) and the substituents on any aryl or hetare 1-3 R^(d);

[0057] het is any mono-, bi-, or tricyclic saturated, unsaturated, oraromatic ring where at least one ring is a 5-, 6- or 7-membered ringcontaining from one to four heteroatoms selected from the groupnitrogen, oxygen, and sulfur, the 5-membered ring having from 0 to 2double bonds and the 6- or 7-membered ring having from 0 to 3 doublebonds and where any carbon or sulfur atoms in the ring may optionally beoxidized, and where any nitrogen heteroatom may optionally bequaternized and where any ring may contain from 0-3 R^(d);

[0058] U is an optionally substituted bivalent radical selected from thegroup; C₁-C₆alkyl, C₀-C₆alkyl-Q, C₂-C₆alkenyl-Q, and C₂-C₆alkynyl-Q,where the substituits on any alkyl, alkenyl or alkynyl are 1-3 R^(a);

[0059] Q is absent or is selected from the group; —O—, —S(O)—, —SO₂—N(Rn)_, —N(R^(n))—, —N(R^(n))—C(═O)—, —N(R^(n))—C(═O)_O_, —N(R^(n))—SO₂—,—C(═O)—, —C(═O)—O—, -het-, —C(═O)₂—N(R^(n))—, —PO(OR^(c))O— and —P(O)O—,where s is 0-2 and the heterocyclic ring is substituted with 0-3 R^(h);

[0060] V is absent or is an optionally substituted bivalent groupselected from C₁-C₆alkyl, C₃-C₈cycloalkyl, C₀-C₆alkyl-C₆-C₁₀aryl, andC₀-C₆alky-het, where the substituits on any alkyl are 1-3 R^(a) and thesubstituents on any aryl or het are 1-3 R^(d);

[0061] W is selected from the group; hydrogen, —OR°, —SR^(m),NR^(n)R^(n′), —NH—C(═O)—O—O—R^(O), —NH—C(═O)—NR^(n)R^(n′),—NH—C(═O)—R^(c), —NH—SO₂—R^(c), —NH—SO₂—NR^(n)R^(n′),—NHSO₂—NH—C(═O)—R^(c), —NH—C(═O)—NH—SO₂—R^(s), —C(═O)—NH—C(═O)—O—R^(O),C(═O)—R^(c), —C(═O)—NH—C(═O)—NR^(n)R^(n′), —C(═O)—NH—SO₂—R^(s),—C(═O)—NH—SO₂—R^(s), SO₂—NH—C(═O)—NR R²—NH—C(═O)—R^(c),—O—C(═O)—NR^(n)R^(n′), —O—C(═O)—R^(c), —O—C(═O)—NH—C(═O)—R^(c),—O—C(═O)—NH—SO₂—R⁵ and —O—SO₂—R^(s);

[0062] Optionally, TBF_(m)-part A together with TBF_(m)-part B andTBF_(n)-part C together with TBF_(n)-part D may independently form Cycle1 substituted with -B-(Cycle 2)-E.

[0063] A drug lead precursor or intermediate of this invention isrepresented by C₀-C₂alkyl-L¹-L²-L³-L⁴-L⁵-C₀-C₂-alkyl where L¹ through L⁵are defined above. Additional embodiments of the present invention willbecome evident to the ordinarily skilled artisan upon a review of thepresent specification.

BRIEF DESCRIPTION OF THE DRAWINGS

[0064]FIG. 1 shows a synthetic reaction wherein an aldehyde is reactedwith O-methyl hydroxylamine to produce an O-methyl oxime compound.

[0065]FIG. 2 shows a variety of organic aldehyde pharmacophore moleculeswhich after conversion to O-methyl oximes were identified as beingcapable of inhibiting the interaction between CD4 and gp120.

[0066]FIG. 3 shows chemistry useful for chemically coupling twoaldehydes via an O,O′-diamino-alkanediol linker to produce bothheterodimeric and homodimeric oxime compounds.

[0067]FIG. 4 shows a variety of aldehyde pharmacophores found to behighly efficient in dimeric form for inhibiting the interaction betweenCD4 and gp120.

[0068]FIG. 5 shows an O-methyl oxime compound found to be particularlyeffective in dimeric form for inhibiting the interaction between CD4 andgp120.

[0069]FIG. 6 shows a specific ligand having particularly high activityfor inhibiting the interaction between CD4 and gp120.

[0070]FIG. 7 shows a synthetic reaction wherein an aldehyde is reactedwith a dimethylamine in the presence of support-boundtriacetoxyborohydride to give rise to an N,N-dimethylamine organiccompound.

[0071]FIG. 8 shows a chemical synthesis sequence resulting in theproduction of a ligand of the present invention.

[0072]FIG. 9 shows a flow diagram for the fragment assembly sequenceresulting in the production of a drug lead compound of the presentinvention.

DETAILED DESCRIPTION OF THE INVENTION

[0073] I. General Description

[0074] The present invention provides a rapid and efficient method foridentifying small molecule candidate target binding fragments or ligandsthat are capable of binding with high affinity to target biologicalmolecules (TBM) of interest. The compounds identified by the subjectmethod find use, for example, as drug lead compounds for the developmentof novel therapeutic drugs. The subject method involves the assembly ofa library of small organic candidate target binding fragments which arecapable of being chemically cross-linked via a linker element. Thelibrary of organic candidate target binding fragments may be“pre-screened” in order to identify members of the library that arecapable of binding to a target biological molecule. At least a portionof the small organic candidate target binding fragments identifiedduring the “pre-screen” as being capable of binding to the target arethen chemically cross-linked to one another in various combinations toprovide a library of potential or candidate cross-linked target bindingfragments for binding to the target biological molecule. The library ofcandidate cross-linked target binding fragments having potential to behigh affinity antagonists (or agonists) are then screened to identifydrug lead compounds that bind to the target biological molecule withhigh affinity. The step of “pre-screening” the library of small organiccandidate target binding fragments to identify those that are capable ofbinding to the target allows one to limit the library of potentialbinding ligands to only those that are comprised of the most favorableorganic candidate target binding fragment building blocks, therebydecreasing the required complexity of the library of potential ligandswhile increasing the probability of identifying a molecule exhibitinghigh binding affinity for the biological target molecule.

[0075] One embodiment of the present invention is directed to a methodfor identifying a drug lead compound that binds to a target biologicalmolecule of interest. The subject method involves assembling a libraryof organic candidate target binding fragment(s) that are capable ofbeing chemically cross-linked via a chemically compatible cross-linkerto provide candidate cross-linked target binding fragment(s) for bindingto the target biological molecule. In this regard, the phrase“assembling a library of organic candidate target binding fragments” isto be construed broadly and is intended to encompass all means by whichone may obtain a library comprising two or more organic compounds whichinclude, for example, obtaining such compounds from a commercial ornon-commercial source, synthesizing such compounds using standardchemical synthesis technology or combinatorial synthesis technology (seeGallop et al. (1994), supra, Gordon et al. (1994), supra, Czarnik andEllman (1996), supra, Thompson and Ellman (1996), supra and Balkenhohlet al. (1996) supra) and obtaining such compounds as degradationproducts from larger precursor compounds, e.g. known therapeutic drugs,large chemical molecules, and the like.

[0076] The candidate target binding fragments (CTBF) and are, for themost part, small water soluble organic molecules that have one or morechemically reactive functionalities, referred to as linkable (orlinkage) functional groups (LFG) (or sites that may be readily convertedto a chemically reactive functionality using standard technology (BLFG))that provide a site for coupling to another compound or candidate targetbinding fragment via a chemically compatible cross-linker. Thus, thecandidate target binding fragments of the present invention are capableof being chemically coupled to one another via a cross-linker to providecandidate cross-linked target binding fragments for binding to thetarget biological molecule, meaning that the candidate target bindingfragment compounds have a reactive functionality, or a site that can bereadily chemically converted to a reactive functionality, where achemically compatible cross-linker may covalently attach thereto,thereby allowing multimerization of the candidate target bindingfragments through the cross-linker. “Ligands”, “candidate ligands' or“candidate cross-linked target binding fragments” for binding to a“target biological molecule” for purposes herein are compounds that areobtained from reacting two or more organic compounds, which may be thesame or different, preferably different, with one or more cross-linkerso as to produce a molecule comprising two or more target bindingfragments and one or more cross-linker. Such ligands are referred toherein as candidate cross-linked target binding fragments (CXL-TBF).

[0077] Candidate target binding fragments having the linkage functionalgroup modified or blocked so that it contains substantially the samelinking group as is found in the candidate cross-linked bindingfragments are sometimes referred to as monomers. Monomers or monomericcompounds that find use in the present invention include, for example,aldehydes, ketones, oximes, such as O-alkyl oximes, preferably O-methyloximes, hydrazones, semicarbazones, carbazides, primary amines,secondary amines, such as N-methylamines, tertiary amines, such asN,N-dimethylamines, N-substituted hydrazines, hydrazides, alcohols,ethers, thiols, thioethers, thioesters, disulfides, carboxylic acids,esters, amides, ureas, carbamates, carbonates, ketals, thioketals,acetals, thioacetals, aryl halides, aryl sulfonates, alkyl halides,alkyl sulfonates, aromatic compounds, heterocyclic compounds, anilines,alkenes, alkynes, diols, amino alcohols, oxazolidines, oxazolines,thiazolidines, thiazolines, enamines, sulfonamides, epoxides, andaziridines, and the like, all of which have chemically reactivefunctionalities (or are directly prepared from precursor compounds thathave chemically reactive functionalities) capable of linking, eitherdirectly or indirectly, to a cross-linker. In fact, virtually any smallorganic molecule that is capable of being chemically coupled to anothersmall organic molecule may find use in the present invention with theproviso that it is sufficiently soluble in aqueous solutions to betested for its ability to bind to a target biological molecule.

[0078] The above described monomers or candidate target bindingfragments will serve as the individual building blocks for candidatecross-linked binding fragments prepared therefrom. Candidate targetbinding fragments that find use herein will generally be less than about2000 daltons in size, usually less than about 1500 daltons in size, moreusually less than about 750 daltons in size, preferably less than about500 daltons in size, often less than about 250 daltons in size and moreoften less than about 200 daltons in size, although organic moleculeslarger than 2000 daltons in size will also find use herein. Candidatetarget binding fragments that find use may be employed in the hereindescribed method as originally obtained from a commercial ornon-commercial source (for example, a large number of small organicchemical compounds that serve as candidate target binding fragments arereadily obtainable from commercial suppliers such as Aldrich ChemicalCo., Milwaukee, Wis. and Sigma Chemical Co., St. Louis, Mo.) or may beobtained by chemical synthesis. Examples of the latter include thepreparation of a library of organic oxime compounds from a single stepcondensation of commercially available aldehydes with O-alkylhydroxylamine as described in Example I below and the preparation of alibrary of N,N-dimethylamine candidate cross-linked target bindingfragments from the reductive amination of commercially availablealdehydes and dimethylamine using support-bound triacetoxyborohydride asdescribed in Example II below and by Kaldor et al., Tetrahedron Lett.37:7193-7196 (1996).

[0079] Libraries of candidate target binding fragments or candidatecross-linked target binding fragments which find use herein willgenerally comprise at least 2 organic compounds, often at least about 25different organic compounds, more often at least about 100 differentorganic compounds, usually at least about 500 different organiccompounds, more usually at least about 1000 different organic compounds,preferably at least about 2500 different organic compounds, morepreferably at least about 5000 different organic compounds and mostpreferably at least about 10,000 or more different organic compounds.Libraries may be selected or constructed such that each individualmolecule of the library may be spatially separated from the othermolecules of the library (e.g., each member of the library is present ina separate microtiter well) or two or more members of the library may becombined if methods for deconvolution are readily available. The methodsby which the library of organic compounds are prepared will not becritical to the invention.

[0080] Once assembled, a library of organic candidate target bindingfragments will be screened using one of any number of different knownassays for the purpose of identifying candidate cross-linked targetbinding fragments that are capable of binding to a target biolocicalmolecule of interest. “Biological target molecules”, “target biologicalmolecules”, “target biomolecules”, “molecular targets”, “biologicaltargets”, and other grammatical equivalents refer to target biologicalmolecules (TBM) that are available (either commercially, recombinantly,synthetically or otherwise) in sufficient quantities for use in in vitrobinding assays and for which there is some interest for identifying ahigh affinity binding partner. For the most part, target biologicalmolecules are proteins, including human proteins or human pathogenproteins that may be associated with a human disease condition, such ascell surface and soluble receptor proteins, such as lymphocyte cellsurface receptors, enzymes, such as proteases, clotting factors,serine/threonine kinases and dephosphorylases, tyrosine kinases anddephosphorylases, bacterial enzymes, fungal enzymes and viral enzymes,signal transduction molecules, transcription factors, proteinsassociated with DNA and/or RNA synthesis or degradation,immunoglobulins, hormones, receptors for various cytokines including,for example, erythropoietin/EPO, granulocyte colony stimulatingreceptor, granulocyte macrophage colony stimulating receptor,thrombopoietin (TPO), IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-10, IL-11,IL-12, growth hormone, prolactin, human placental lactogen (LPL), CNTF,octostatin, various chemokines and their receptors such as RANTES,MIP1-α, IL-8, various ligands and receptors for tyrosine kinases such asinsulin, insulin-like growth factor 1 (IGF-1), epidermal growth factor(EGF), heregulin-α and heregulin-β, vascular endothelial growth factor(VEGF), placental growth factor (PLGF), tissue growth factors (TGF-α andTGF-β), other hormones and receptors such as bone morphogenic factors,folical stimulating hormone (FSH), and leutinizing hormone (LH), tissuenecrosis factor (TNF), apoptosis factor-1 and -2 (AP-1 and AP-2), andproteins and receptors that share 20% or more sequence identity tothese, and the like, nucleic acids, including both DNA and RNA,saccharide complexes, and the like.

[0081] For the step(s) of screening libraries of candidate targetbinding fragments or candidate cross-linked target binding fragments formembers having the ability to bind to a target biological molecule ofinterest, a simple ELISA assay may be used to (a) identify member(s) ofthe library that are capable of binding to the target, and (b) determinethe approximate K_(d) with which the library member(s) bind to themolecular target. While ELISA assays are preferred for screeninglibraries of organic compounds, virtually any in vitro assay that allowsone to detect binding of the target biological molecule by an organiccompound may be employed for screening the library, wherein such assaysinclude ELISA, other sandwich-type binding assays, binding assays whichemploy labeled molecules such as radioactively or fluorescently labeledmolecules, fluorescence depolarization, calorimetry, proteindenaturation, resistance to proteolysis, gel filtration, equilibriumdialysis, surface plasmin resonance, X-ray crystallography, and thelike. Such assays either measure the ability of library members to binddirectly to the target biological molecule or are competition bindingassays designed to measure the ability of library members to inhibit theinteraction between the target biological molecule and another moleculethat binds to the target biological molecule. Any of the above assaysmay be employed to screen libraries of candidate compounds to identifythose that bind to a target biological molecule.

[0082] For the step of screening a library of candidate compounds toidentify those that bind to a target biological molecule, it will bewell within the skill level in the art to determine the concentration ofthe library members to be employed in the binding assay. For the mostpart, the screening assays will employ concentrations of candidatecompounds ranging from about 0.01 to 10 mM, preferably from about 0.05to 5 mM.

[0083] The step of pre-screening a library of candidate target bindingfragments to identify those that bind to a target biological moleculeallows one to identify and isolate only those members of the librarythat have some binding affinity for the target. As such, in contrast tostandard combinatorial library approaches, the small organic buildingblocks are “pre-screened” to select a smaller set of compounds that havesome binding affinity for the target. Thus, the most productive organiccompound building blocks can be identified for incorporation into thepotential high affinity candidate cross-linked target binding fragmentsthat are prepared therefrom, without having to screen all possiblecombinations of all of the initial candidate target binding fragmentbuilding blocks. Generally, the candidate target binding fragmentlibrary members selected as building blocks for subsequently preparedcandidate cross-linked target binding fragments are those that have thehighest affinity for binding to the target biological molecule. For themost part, candidate target binding fragments chosen as building blocksfor incorporation into the subsequently prepared candidate cross-linkedtarget binding fragments are those that bind to the target biologicalmolecule with a K_(d) of about 10 mM or less, usually about 5 mM orless, more usually about 1 mM or less, preferably about 500 μM or less,more preferably about 100 μM or less and most preferably about 50 μM orless. However, for some applications, one or more of the candidatetarget binding fragment(s) chosen for incorporation into thesubsequently prepared candidate cross-linked target binding fragmentsmay have an individual K_(d) for the target biological molecule ofgreater than 10 mM.

[0084] Once candidate target binding fragments that bind to a targetbiological molecule with some desired degree of affinity are identified,at least a portion of those compounds (or structurally related analogsthereof) are chemically coupled via a cross-linker to provide a libraryof candidate cross-linked target binding fragments for binding to thetarget biological molecule, wherein those candidate cross-linked targetbinding fragments comprise at least one candidate target bindingfragment reacted or linked with a cross-linker. Usually two or morecandidate target binding fragments (or structurally related analogsthereof) linked by a cross-linker are combined and in some cases thesetwo fragments are the same. The two (or more) candidate target bindingfragments (or structurally related analogs thereof) incorporated into acandidate cross-linked target binding fragment may be the same (i.e., toprovide a homodimer or homomultimer) or different (i.e., to provide aheterodimer or heteromultimer). Most commonly, the two candidate targetbinding fragments in the candidate cross-linked target binding fragmentare different.

[0085] By “structurally related analog”, “analog” and the like, is meanta fragment compound that has the same chemical structure as a fragmentidentified as being capable of binding to the target biological moleculeexcept that the analog has a different chemically reactive functionalityor linkage functional group (LFG) for binding to the cross-linker thandoes the fragment that was identified as being capable of binding to thetarget biological molecule in the first or pre-screen. The analog mayalso optionally possess or lack one or more substituents that are eitherlacking or present, respectively, on the fragments identified in thepre-screening provided that the presence or absence of thosesubstituents does not substantially alter the compounds ability to bindto the target. As such, while one may pre-screen a library of, forexample, candidate oxime compounds to identify candidate oxime fragmentsthat bind to the target biological molecule, one can chemically couplenot the actual oxime compounds identified in the “pre-screening” butrather aldehydes having that same chemical structures as the oximesidentified in the screen (but which have an aldehyde reactivefunctionality rather than an oxime reactive functionality). The presentinvention, therefore, encompasses not only chemical coupling of theactual compounds identified in the initial “pre-screening step” (e.g.,aldehydes are prescreened and also subsequently linked), but also thechemical coupling of structurally related analogs of those compounds(e.g., oximes are pre-screened but the analogous aldehydes are linked).

[0086] As described above, candidate target binding fragment willcomprise a chemically reactive functionality or linkage functional group(LFG) (or a site that can be converted to a chemically reactivefunctionality (BLFG)) to which a cross-linker may be covalently bound,thereby providing a means for cross-linking two or more candidate targetbinding fragments having a LFG (or analog or blocked form thereof) toprovide a candidate cross-linked target binding fragment. Therefore,cross-linkers that find use herein will be multifunctional, preferablybifunctional, crosslinking molecules that can function to covalentlybond at least two fragment compounds together via their reactivefunctionalities or LFG's. Linkers or cross-linkers (XL) will have atleast one, two or more and preferably only two, chemicallycross-reactive functional groups (CFG) on the cross-linker (XL). Thechemically cross-reactive functional groups that are available forbonding to two or more candidate target binding fragments, wherein thosefunctional groups may appear anywhere on the cross-linker, preferably ateach end of the cross-linker and wherein those chemically cross-reactivefunctional groups may be the same or different depending upon whetherthe candidate target binding fragment is to be linked have the same ordifferent chemically cross-reactive functional groups. Cross-linkersthat find use herein may be substituted or unsubstituted straight-chainor branched alkyl, aryl, alkaryl, heteroaryl, heterocycle and the like.Preferably straight chain alkyl will generally be at least one methylenein length, more generally from 2 to 8 methylenes in length, andoptionally as many as about 12 or more methylenes or the equivalent inlength. Cross-linkers may include atoms or groups to increase or improvesolubility of the library members, such as oxygen atoms interspersedbetween methylene groups creating ether or polyether linkers.Cross-linkers will generally comprise alkyl groups either saturated orunsaturated, and therefore, may comprise alkanes, alkenes or alkynes.Heteroatoms including nitrogen, sulfur, oxygen, and the like may also beappended to the alkyl to form groups such as; alkoxyl, hydroxyalkyl orhydroxy groups. Other cross-linking groups such as aryl, especiallyphenylene or substituted phenylene linkers are suitably employed.Usually cross-linker elements will be of varying lengths, therebyproviding a means for optimizing the binding properties of across-linked target binding fragment prepared therefrom.

[0087] In particularly preferred embodiments, cross-linkers may beO,O′-diamino-alkanediol compounds, preferablyO,O′-diamino-C₁-C₈alkanediol, which are useful for chemically couplingaldehyde organic compounds, or any of a variety of different diaminecompounds, which are useful for chemically coupling aldehyde containingcompounds.

[0088] Various chemistries may be employed for chemically couplingcandidate target binding fragments via a cross-linker to providecandidate cross-linked target binding fragments for binding to a targetbiological molecule. For example, many well known chemistries that canbe employed for chemically coupling candidate target bindingfragments(s) via a linker to form candidate cross-linked target bindingfragments include, for example, reductive aminations between aldehydesand ketones and amines (March, Advanced Organic Chemistry, John Wiley &Sons, New York, 4th edition, 1992, pp.898-900), alternative methods forpreparing amines (March et al., supra, p.1276), reactions betweenaldehydes and ketones and hydrazine derivatives to give hydrazones andhydrazone derivatives such as semicarbazones (March et al., supra,pp.904-906), amide bond formation (March et al., supra, p.1275),formation of ureas (March et al., supra, p.4299), formation ofthiocarbamates (March et al., supra, p.892), formation of carbamates(March et al., supra, p.1280), formation of sulfonamides (March et al.,supra, p.1296), formation of thioethers (March et al., supra, p.1297),formation of disulfides (March et al., supra, p.1284), formation ofethers (March et al., supra, p.1285), formation of esters (March et al.,supra, p.1281), additions to epoxides (March et al., supra, p.368),additions to aziridines (March et al., supra, p.368), formation ofacetals and ketals (March et al., supra, p.1269), formation ofcarbonates (March et al., supra, p.392), formation of enamines (March etal., supra, p.1284), metathesis of alkenes (March et al., supra,pp.1146-1148 and Grubbs et al., Acc. Chem. Res. 28:446-452 (1995)),transition metal-catalyzed couplings of aryl halides and sulfonates withalkenes and acetylenes (e.g., Heck reactions) (March et al., supra,pp.717-178), the reaction of aryl halides and sulfonates withorganometallic reagents (March et al., supra, p.662), such asorganoboron (Miyaura et al., Chem. Rev., 95:2457 (1995)), organotin, andorganozinc reagents, formation of oxazolidines (Ede et al., TetrahedronLetts. 38:7119-7122 (1997)), formation of thiazolidines (Patek et al.,Tetrahedron Letts. 36:2227-2230 (1995)), amines linked through amidinegroups by coupling amines through imidoesters (Davies et al., CanadianJ. Biochem. 50:416422 (1972)), and the like.

[0089] The step of chemically cross-linking, via a cross-linker, atleast a portion of the candidate target binding fragments identified asdescribed above as being capable of binding to the target biologicalmolecule or structurally related analogs thereof provides a library ofcandidate cross-linked target binding fragments for binding to thetarget molecule that comprise at least two of the candidate targetbinding fragments or analogs thereof and the cross-linker. As previouslystated the candidate target binding fragments incorporated intocandidate cross-linked target binding fragments may be the same, therebyproviding a homomultimer, or different, thereby providing aheteromultimer, and libraries of candidate cross-linked target bindingfragments generally comprise both homo- and hetero-multimers. Candidatecross-linked target binding fragments for binding to the target moleculeare preferably dimeric, however, candidate cross-linked target bindingfragments that find use may also be trimeric, tetrameric, and the like,those compounds being obtained by employing cross-linkers having morethan two chemically cross-reactive functional groups for cross-linkingpurposes. Candidate cross-linked target binding fragments for binding toa target biological molecule that find use herein will generally be lessthan about 1000 daltons in size and often less than about 750 daltons insize.

[0090] Libraries of candidate cross-linked target binding fragments forbinding to the target biological molecule will generally comprise atleast 1 candidate cross-linked target binding fragment, usually at leastabout 20 different candidates, more usually at least about 100 differentcandidates, preferably at least about 200 different candidates, morepreferably at least about 500 different candidates, most preferably atleast 1,000 different candidates and often 10,000 or more. Libraries ofcandidate cross-linked target binding fragments may be constructed suchthat each individual molecule of the library may be spatially separatedfrom the other molecules of the library (e.g., each member of thelibrary is in a separate microtiter well) or two or more members of thelibrary may be physically combined if methods for deconvolution arereadily available.

[0091] Once obtained, libraries of candidate cross-linked target bindingfragments for binding to the target biological molecule will be screenedfor the purpose of identifying a member(s) of the library that is/arecapable of binding to the target biological molecule with high affinity.For such purposes, any of the above described screening assays can beemployed, wherein preferably a biological assay such as an ELISA assayis employed.

[0092] For the step of screening a library of candidate cross-linkedtarget binding fragments to identify one or more that bind to a targetbiological molecule, it will be well within the skill level in the artto determine the concentration of the compounds to be employed in thebinding assay. We have herein found that candidate cross-linked targetbinding fragments generated by chemically coupling organic compoundsthat bind to a target biological molecule often exhibit surprisinglyhigh binding affinities for the target. For the most part, candidatecross-linked target binding fragments that serve as potential drug leadcompounds or may have therapeutic efficacy on their own bind to thetarget biological molecules with a K_(d) of about 500 nM or less,usually about 100 nM or less, more usually about 50 nM or less. However,for various applications, one or more drug lead compound(s) having anindividual K_(d) for the target biological molecule of greater than 500nM may also find use.

[0093] Another embodiment of the present invention is directed to amethod for inhibiting the interaction between first and secondbiological molecules which bind to each other, wherein the methodcomprises contacting a system comprising those molecules with a bindinginhibitory amount of a candidate cross-linked target binding fragment ordrug lead compound identified by the method described above, wherein thedrug lead compound or cross-linked target binding fragment binds to thefirst biological molecule and inhibits its ability to bind to the secondbiological molecule. For the most part, the first and second biologicalmolecules will be proteins, nucleic acids, saccharide complexes, and thelike, preferably at least one being a protein, more preferably bothbeing proteins. In particularly preferred embodiments, the first orsecond biological molecule may be CD4 or gp120. In other preferredembodiments, the first biological molecule may be a protein wherein thesecond biological molecule is a receptor for that protein, a nucleicacid, either DNA or RNA, that binds to that protein or a polysaccharideor the first biological molecule is an enzyme wherein the secondbiological molecule is a substrate for that enzyme.

[0094] Systems that comprise both the first and second biologicalmolecules may be either in vitro or in vivo, wherein the first andsecond biological molecules are situated such that they are capable ofbinding to one another. For in vivo applications, the lead of interestmay be administered on its own or in pharmaceutically acceptable media,for example normal saline, PBS, etc. The additives may includebactericidal agents, stabilizers, buffers, or the like. In order toenhance the half-life of the drug lead compounds in vivo, the compoundsmay be encapsulated, introduced into the lumen of liposomes, prepared ascolloids, or another conventional technique may be employed thatprovides for an extended lifetime thereof.

[0095] The drug lead compounds may be administered as a combinationtherapy with other pharmacologically active agents or may physicallylinked to such agents or other carriers. Various methods foradministration may be employed. The drug lead compounds may be givenorally, or may be injected intravascularly, subcutaneously,peritoneally, etc. A “binding inhibitory amount” of the drug leadcompounds will vary widely, depending upon the nature of the first andsecond biological molecules, the frequency of administration, the mannerof administration, the clearance of the compound from the host, and thelike. Appropriate binding inhibitory amounts may be determinedempirically by those skilled in the art in a routine manner candidatecross-linked target binding fragments

[0096] II. Definitions and Preferred Embodiments

[0097] In its broadest embodiment, the method of this inventioncomprises: assembling a library of candidate target binding fragments;screening the library of candidate target binding fragments for thosethat bind to a target molecule; crosslinking target binding fragments toproduce a library of candidate cross-linked target binding fragments;screening the library of candidate cross-linked target binding fragmentsfor those that bind to the target molecule. The product of this methodis referred to as a lead pharmaceutical or drug candidate.

[0098] More specifically (see FIG. 9), the method of this invention isused to identify lead pharmaceutical drug candidates and optionallyinvolves the following simple steps (a-h).

[0099] (a) Assembling a Library of Candidate Target Binding Fragmentseach Fragment Having a Linkable (or Linkage) Functional Group (LFG) orthe Blocked Form Thereof (BLFG), the Blocked Form Containing LinkingGroup (LG);

[0100] Assembling a library as used herein means any method of selectingtwo or more molecules to form a library for use in the method of thisinvention. Preferably the library will be large, greater than 50members, and contain a diverse array of target interactive groupscapable of forming non-covalent bonds, e.g. hydrogen, Van der Waals,electrostatic, hydrophobic and the like with the target molecule. Suchinteractive groups may include functional groups found on naturallyoccurring amino acid sidechains, carbohydrates, lipids, nucleic acidsand their metabolites and derivatives thereof, or groups found on knownpharmaceuticals. Optionally, a library may be customized to containinteractive groups known or suspected to interact with binding sites onthe biological target molecule or its biological ligand.

[0101] Candidate target binding fragments (CTBF) are small water solubleorganic molecules having a molecular weight of about 200 da (includingthe LFG) capable of forming a non-covalent complex with a targetbiological molecule (TBM). This complex may be of low affinity having aK_(d) as low as about 5 mM. The CTBF's are commercially available or maybe synthesized by known procedures.

[0102] Linkable (or linkage) functional groups (LFG) include anyfunctional groups capable of reacting with a chemically cross-reactivefunctional group (CFG) on a cross-linker (XL) thereby forming a stablecovalent bond with the cross-linker. This covalent bond is referred tosimply as a linking group (LG). When the linked molecule contains morethan one linking group an integer following LG is used to indicate thenumber of the LG in the molecule. The LFG includes blocked, protected orotherwise transformed groups that may or may not react directly with theCFG on the cross-linker. This blocked form of the linkable functionalgroup (BLFG) is often the preferred form of the CTBF because it may beless likely to form covalent linkages with the target biologicalmolecule in the contacting step below.

[0103] In the case where the BLFG is to be used in the contacting step,the de-protected or re-transformed form of the CTBF that it is capableor reacting with the CFG on the cross-linker is the form used in thecross-linking step below. By way of illustration, a CTBF may contain analdehyde as a LFG or this aldehyde may be protected or transformed byreacting it with an O-amino alcohol to form an oxime (BLFG) as shownbelow.

[0104] In this case the oxime would be considered the BLFG. It will beappreciated that more than one reaction or transformation to an LFG maybe made. By way of further illustration an aldehyde may be reacted withan amine to form a Schiff base, which in turn may be reduced to asecondary amine. This may be still further reacted to form an amide,sulfonamide, urea, carbamate etc. All these transformations of theinitial aldehyde are also considered BLFG's.

[0105] In the case where the CTBF (including the LFG) is firsttransformed or protected, the initial CTBF is sometimes referred to as a“precursor” while the transformed or protected form that is contactedwith the TBM is referred to as a “monomer”. Thus in the reactionillustrated above, the aldehyde may be called a precursor while theoxime is referred to as a monomer. In this case the oxime covalent bond(═N—O—) is referred to as the linking group (LG). It is often preferredthat the monomer contain the same linking group (LG) as is present inthe cross-linked form, described below, because some of the bindingenergy with the target may come from LG.

[0106] Examples of linkage or linkable functional groups include;aldehyde, ketone, primary amine, secondary amine, epoxide, carboxylicacid, sulfonic acid, alcohol (hydroxyl), isocyanate, isothiocyanate,halide and sulfonate. These functional groups may act as precursors ofthe blocked linkage functional groups.

[0107] Examples of CTBF's having blocked linkage or linkable functionalgroups are molecules containing Linkage Groups selected from; oxime,hydrazone, N-acyl hydrazone, secondary amine, tertiary amine, acetal,ketal, 1,2 amino alcohols, amide, N,N-disubstituted amides, thioamide,ureido, thioureido, carbamate, thiocarbamate, thiothiocarbamate,sulfonamide, carbamate, guanidino, amidino, thioester, ester, ether,2-hydroxyether, 2-hydroxythioether, thioether, disulfide, alkane(alkylene), alkene (alkenylene) and alkyne (alkynylene). Preferredmonomers will contain the above functional groups as LG's.

[0108] Steps b-d below may be combined into a single screening step thatmay be referred to as a first screening step, a pre-selection step or apre-screening step.

[0109] (b) Contacting the Candidate Target Binding Fragments with aTarget Biological Molecule (TBM).

[0110] Contacting of the TBM with one or more members of the library maybe conducted either individually or multiply. Preferably each candidatetarget binding fragment is contacted individually with the TBM. Forexample, this may conveniently be done in a 96 well format plate so thatthe formation of a complex with each member of the library can beconveniently evaluated without requiring any deconvolution step. Thecontacting step is often conducted at relatively high concentrations ofthe CTBF, so that K_(d)'s as low as about 5 mM can be measured (seebelow).

[0111] The Target Biological Molecule (TBM) may be any biologicalmolecule preferably of mammalian and most preferably of human origin.Optionally preferred TBM's may be human pathogen proteins such as viralproteins from viruses that infect human cells. Preferred TBM's areproteins most preferably secreted proteins. Preferred secreted proteinsinclude; enzymes, cytokines, hormones, growth factors and theirreceptors. The TBM's may be isolated from natural sources or maderecombinately in a host cell. Normally the atoms of the TBM will containthe natural abundance isotopes, but in some circumstances may beenriched. When the target is a receptor or a cell surface boundmolecule, the TBM may conveniently be the extracellular domain or aderivative thereof.

[0112] In a preferred embodiment of the method, the Target BiologicalMolecule is not a single biological molecule; rather it is two or moremolecules. For example, preferred TBM's may be protein-protein,protein-DNA/RNA, protein-substrate pairs. By way of illustration, aligand-receptor pair may be the actual target when an ELISA or otherbiological assay requiring two or more biological molecules is used tomeasure a physical association (see below) such as binding or activity.The binding constant, IC50 or other measurement may result from the CTBFbinding with either the Ligand, receptor or both. Similarly, whenselected fragments are cross-linked (see below) and re-screened againstthe ligand-receptor pair, the recombined fragments may bind with theligand, receptor or both.

[0113] (c) Measuring a change in a first physical association (PA-1) ofthe target biological molecule.

[0114] Measuring a change as used herein means any method capable ofquantifying a physical association, including binding, of the CTBF withthe TBM.

[0115] A physical association (PA-1) of the target molecule includes abiological property such as binding with another biological molecule,signal transduction or catalysis of a reaction. It may also include anymeasurable physical chemical property such as a spectroscopic ormagnetic property. Preferably the physical association will be suitablefor rapid high throughput screening. The most preferred physicalassociation measurement will be a biological one such as measuringbinding or catalysis. An example of binding measurement would be anELISA assay where protein-protein antagonism is measured.

[0116] (d) Selecting target binding fragments (TBF) based on (c).

[0117] Selecting target binding fragments (TBF) is based on the physicalassociation measurement step. Selected TBF's will include those thatbind relatively weakly with the TBM. Thus, for example in a bindingassay such as ELISA, fragments that bind with no greater than a 5 mMaffinity may be selected for. Most commonly the first selected CTBF'swill bind to the target with an affinity of from 2 mM to 100 μM. TBF'sor monomers that bind with a higher affinity e.g. K_(d)<50 μM arepreferred, however such relatively high binding affinities are notnecessary for selection for the cross-linking step.

[0118] (e) Reacting target binding fragments with a cross-linker, havingchemically compatible cross-reactive group(s) with the LFG, underconditions suitable for forming a library of candidate cross-linkedtarget binding fragments (CXL-TBF).

[0119] The selected target binding fragments, which individuallynormally bind to the target with relatively low affinity, are thencross-linked, normally in all combinations and permutations, to producea library of candidate cross-linked target binding fragments (CXL-TBF).By way or illustration, If TBF_(m) and TBF_(n) are selected from thefirst screen, these molecules are then reacted with a suitablecross-linker such as a bifunctional linker (BFL) to form candidatecross-linked target binding fragments (CXL-TBF) for the second screeningstep according to the general equation:

[0120] The BFL above will normally have two cross-reactive groups thatare compatible with both LFG's, that is they will form stable covalentbond(s) (LG) with the LFG's under selected reaction conditions.

[0121] Each TBF may consist of two (or three) parts or moieties when LFGcontains an atom capable of forming two (or three) bonds other than thelinking group (LG) bond formed with the cross-linker. For example, whenLFG is the carbonyl of a ketone, the carbonyl carbon may be bonded totwo alkyl, aryl etc parts or moieties (part A and part B or part C andpart D). This case may be represented generally by the diagram belowwhere, for example, two ketones are cross-linked with anO,O′-diamino-alkanediol cross-linker to form a di-oxime:

[0122] Here each of the two alkyl, aryl etc parts or moieties from theketone are bonded to a nitrogen atom in the linking group (LG, ═N—O—).

[0123] The CXL-TBF's are sometimes referred to as “dimers”, however,they are truly only dimers when the cross-linking moiety (XL) is only achemical bond. In the most general case these “dimers” will contain alinking moiety, such as an alkane, alkene, arylene, alkyl ether and thelike bonded through one or more linking groups (LG) to the correspondingTMF's.

[0124] Reacting means chemically reacting so that a stable covalent bondor linking group LG is formed as the reaction product between thelinkable functional group on the target binding fragment(s) and achemically cross-reactive functional group (CFG) on the cross-linker.This step may be referred to as a cross-linking step or sometimes a“combination” or “recombination” step.

[0125] Compatible functional group herein means capable of reacting withto form a stable LG.

[0126] The cross-linker is a small organic molecule having at least onecross-reactive functional group capable of reacting with the linkablefunctional group of at least one of the target binding fragments.Commonly, the cross-linker will have 24 such cross-reactive functionalgroups and most commonly 2 such groups. In this case the cross-linker isreferred to as a bifunctional linker (BFL), either homo-bifunctional orhetero-bifunctional depending on whether the compatible functionalgroups are the same or different from one another.

[0127] In the case where the cross-linker has a single cross-reactivefunctional group, the candidate cross-linked target binding fragment,CKL-TBF, is simply the reaction product of these two molecules:

[0128] A simple example of such a reaction would be the product of analdehyde and an amine. In this exemplary case R1 is a TBF and R2 Is XLwhere LFG-1 is an aldehyde and CFG-1 is a primary amine:

[0129] In this case LG is the resulting secondary amine, —(NH)—.

[0130] In the case where the cross-linker is a bifunctional linker, BFL,the linker has two cross-reactive linking groups and may be representedgenerally as:

[0131] In this case, reacting two target binding fragments from thefirst screen above, each with its own linking functional group with theabove BFL may be represented:

[0132] In the most general case of the above reaction, the two targetbinding fragments will have different linkable functional groups andwill be linked by a hetero-bifunctional linker (het-BFL). In the mostcommon case, both TBF will have the same CFG and will be linked by ahomo-bifunctional linker (homo-BFL). In some cases, target bindingfragments having different linkable functional groups may be linked witha homo-BFL and target binding fragments having the same linkablefunctional groups may be linked with a het-BFL.

[0133] Higher order linking groups such as tri- and tetra-functionallinkers may also be employed in the method of this invention. By way ofillustration a trifunctional linker such as that shown below may beused:

[0134] In each of the above cases, the product of the cross-linkingreaction produces a candidate cross-linked target binding fragment(s)(CXL-TBF). These candidate molecules, typically with a molecular weightof 400-600 daltons, are then screened a second time against the targetbiological molecule.

[0135] Steps f-h below may be combined into a single screening step thatmay be referred to as a second screening step, a selection step or afinal screening step.

[0136] (f) Contacting the Candidate Cross-Linked Target BindingFragments with the Target Biological Molecule.

[0137] Contacting of the TBM and one or more members of the library ofcandidate cross-linked target binding fragments (CXL-TBF) may beconducted either individually or multiply as before. Preferably eachcandidate cross-linked target binding fragment is contacted individuallywith the TBM. The contacting step is usually conducted at lowerconcentrations of the CXL-TBF compared to CTBF's in the first screeningstep, so that K_(d)'s in the micromolar or nanomolar range can bemeasured. The contacting format may be the same or different from thatin the first or pre-screening step. Thus for example the two contactingsteps may both be part of a binding assay (e.g. ELISA) or, for example,the second contacting step may be a functional activity assay or cellbased binding assay.

[0138] (g) Measuring a Change in a Second Physical Association (PA-2) ofthe Target Biological Molecule.

[0139] The second physical association (PA-2) measurement for theCXL-TBF's may be the same as that used for the CTBF's or may be adifferent physical measurement. Preferably the second physicalassociation measurement will be a biological measurement rather than aphysical chemical measurement such as spectroscopic or the like.

[0140] (h) Selecting Cross-Linked Target Binding Fragments (XL-TBF)Based of (g).

[0141] The cross-linked target binding fragments (XL-TBF) selected willtypically have a K_(d), IC-50 or the equivalent of 500 nM or better.These XL-TBF's are useful as drug lead pharmaceutical candidatemolecules.

[0142] III Specific Chemistry

[0143] Many chemistries may be employed to produce linkable functionalgroups or to block or derivitize them. Similarly a wide array or linkingchemistries are possible. Described below are a number of chemistriessuitable for rapid high throughput screening. This description is meantto be illustrative and not limiting. In the description below the term“CTBF” is equivalent to “CTBF-part A” plus “CTBF-part B” defined above.Similarly, Linkable functional group is used interchangeably withlinkage functional group.

[0144] The term “alkyl” means a cyclic, branched or unbranched saturatedor unsaturated hydrocarbon radical having the number of carbonsspecified, or if no number is specified, up to 12 carbon atoms.

[0145] The term “aryl” means a homocyclic aromatic hydrocarbon radicalhaving from 6-14 carbon atoms. Examples include phenyl, napthyl,biphenyl, phenanthrenyl, napthacenyl and the like.

[0146] The term “heteroaryl” means a heterocyclic aromatic radicalhaving from 4-13 carbon atoms and from 1-6 heteroatoms selected from N,O, S and P.

[0147] The term “heterocycle” means a saturated or partially unsaturatedcyclic radical having from 3-13 carbon atoms and from 1-6 heteroatomsselected from O, S, N and P.

[0148] The term “alkoxy” means an alkyl radical, as defined above,substituted with an oxo radical (—O—).

[0149] The term “acyl” means alkanoyl or alkylcarbonyl having from 1-12carbon atoms.

[0150] The term “carboxy ester” means acyloxy or alkanoyloxy having from1-12 carbon atoms.

[0151] The term “carboxamide” means alkylcarbonylamino having from 1-12carbon atoms.

[0152] GENERAL NOTE: When CTBFs are employed with BLFGs, they may beprepared as described below from CTBFs containing the correspondingLFGs. Alternatively, it may be practical to purchase, otherwise acquire,or prepare by known methods the CTBFs with BLFGs using alternativechemistry.

Candidate Target Binding Fragments or Molecules (CTBFs)

[0153] I. Aldehyde and Ketone as the Linkage Functional Groups and theCorresponding Blocked Linkage Functional Groups

[0154] (a). The linkage functional group is the carbonyl group presentin an aldehyde or a ketone. These CTBFs may be available commercially,or may be prepared by a variety of known methods to those practiced inthe art. Aldehyde CTBFs are represented as follows.

[0155] Ketone CTBFs are represented with two different parts (A and B)of the CTBF attached to ketone carbonyl as shown below.

[0156] (b). Alternatively, the library is assembled with the oxime groupas the blocked linkage functional group (BLFG). These CTBF's areprepared by condensation of aldehydes or ketones LFG's with anO-substituted hydroxylamine. R⁸ may be H, or a straight chain orbranched alkyl group of length 1 to 10, which may incorporate from 1 to10 heteroatoms (N, O, P, S) within the chain. R⁸ may also be appendedwith up to five R⁹ groups (R⁹ is alkyl, aryl, heteroaryl, heterocycle,carboxy ester, carboxamide, amino, N-acylamino, alkoxy, hydroxy,mercapto, phosphono, sulphono). R⁸ may also be an aryl or heteroarylgroup that is optionally substituted (alkyl, aryl, heteroaryl, carboxyester, carboxamide, amino, N-acylamino, alkoxy, hydroxy, mercapto,phosphono).

[0157] (c). Alternatively, the library is assembled with the hydrazonegroup as the BLFG. These CTBFs are prepared by condensation of aldehydesor ketones LFGs with an N-substituted hydrazine (March, Advanced OrganicChemistry, John Wiley & Sons, New York, 4^(th) edition, 1992, pp.904-906). R⁸ may be H, or a straight chain or branched alkyl group oflength 1 to 10, which may incorporate from 1 to 10 heteroatoms (N, O, P,S) within the chain. R⁸ may also be appended with up to five R⁹ groups(R⁹ is alkyl, aryl, heteroaryl, carboxy ester, carboxamide, amino,N-acylamino, alkoxy, hydroxy, mercapto, phosphono, sulphono). R⁸ mayalso be an aryl or heteroaryl group that is optionally substituted(alkyl, aryl, heteroaryl, carboxy ester, carboxamide, amino,N-acylamino, alkoxy, hydroxy, mercapto, phosphono).

[0158] (d). Alternatively, the library is assembled with theN-acylhydrazone group as the BLFG. These CTBF's are prepared bycondensation of aldehydes or ketone LFG's and an N-acyl hydrazine. Manyreaction conditions are known to those practiced in the art (e.g.,[March et al. supra, pp. 905-906] and [Li et al. Chem. Biol., 1: 37(1994)]). X may be nothing, O, S, NH or NR⁹. R⁸ or R⁹ may be H, or astraight chain or branched alkyl group of length 1 to 10, which mayincorporate from 1 to 10 heteroatoms (N, O, P, S) within the chain. R⁸or R⁹ may also be appended with up to five R¹⁰ groups (R¹⁰ is alkyl,aryl, heteroaryl, carboxy ester, carboxamide, amino, N₁₀ acylamino,alkoxy, hydroxy, mercapto, phosphono, sulphono). R⁸ or R⁹ may also be anaryl or heteroaryl group that is optionally substituted (alkyl, aryl,heteroaryl, carboxy ester, carboxamide, amino, N-acylamino, alkoxy,hydroxy, mercapto, phosphono).

[0159] (e). Alternatively, the library is assembled with an amine groupas the BLFG. These CTBF's are prepared by reductive amination ofaldehydes or ketone LFGs. A large number of reducing agents could beemployed that are known to those practiced in the art (March, et al.supra, pp. 898-900). R⁸ or R⁹ may be H, or a straight chain or branchedalkyl group of length 1 to 10, which may incorporate from 1 to 10heteroatoms (N, O, P, S) within the chain. R⁸ or R⁹ may also be appendedwith up to five R¹⁰ groups (R¹⁰ is alkyl, aryl, heteroaryl, carboxyester, carboxamide, amino, N-acylamino, alkoxy, hydroxy, mercapto,phosphono, sulphono). R⁸ or R⁹ may also be an aryl or heteroaryl groupthat is optionally substituted (alkyl, aryl, heteroaryl, carboxy ester,carboxamide, amino, N-acylamino, alkoxy, hydroxy, mercapto, phosphono).

[0160] (f). Alternatively, the library is assembled with an acetal orketal group as the BLFG. These CTBF's are prepared by condensing thealdehyde or ketone LFG's with a diol. Conditions for the preparation ofacetals or ketals are known to those practiced in the art (March, et al.supra, pp. 889-891). R⁸ may be a straight chain or branched alkyl groupof length 2 to 10, which may incorporate from 1 to 10 heteroatoms (N, O,P, S) within the chain. R⁸ may also be appended with up to five R⁹groups (R⁹ is alkyl, aryl, heteroaryl, carboxy ester, carboxamide,amino, N-acylamino, alkoxy, hydroxy, mercapto, phosphono, sulphono). R⁸may also be an aryl or heteroaryl group that is optionally substituted(alkyl, aryl, heteroaryl, carboxy ester, carboxamide, amino,N-acylamino, alkoxy, hydroxy, mercapto, phosphono). R⁸ may be a straightchain or branched alkyl group of length 2 to 10, which may incorporatefrom 1 to 10 heteroatoms (N, O, P, S) within the chain. R⁸ may also beappended with up to five R⁹ groups (R⁹ is alkyl, aryl, heteroaryl,carboxy ester, carboxamide, amino, N-acylamino, alkoxy, hydroxy,mercapto, phosphono, sulphono). R⁸ may also be an aryl or heteroarylgroup that is optionally substituted (alkyl, aryl, heteroaryl, carboxyester, carboxamide, amino, N-acylamino, alkoxy, hydroxy, mercapto,phosphono).

[0161] (g). Alternatively, the library BLFG's may be prepared bycondensing the aldehyde or ketone LFGS with an amino alcohol or an aminothiol. Methods to prepare the product oxazolidines and thiazolidines areknown to those practiced in the art (e.g., oxazolidines: Ede, et al.Tetrahedron Letters, 38: 7119-7122 (1997), and thiazolidines: Patek etal. Tetrahedron Letters, 36:2227-2230 (1995)). R⁸ or may be a straightchain or branched alkyl group of length 2 to 10, which may incorporatefrom 1 to 10 heteroatoms (N, O, P, S) within the chain. R⁸ may also beappended with up to five R¹⁰ groups (R¹⁰ is alkyl, aryl, heteroaryl,carboxy ester, carboxamide, amino, N-acylamino, alkoxy, hydroxy,mercapto, phosphono, sulphono). R⁸ may also be an aryl or heteroarylgroup that is optionally substituted (alkyl, aryl, heteroaryl, carboxyester, carboxamide, amino, N-acylamino, alkoxy, hydroxy, mercapto,phosphono). R⁹ may be H, or a straight chain or branched alkyl group oflength 1 to 10, which may incorporate from 1 to 10 heteroatoms (N, O, P,S) within the chain. R⁹ may also be appended with up to five R¹¹ groups(R¹¹ is alkyl, aryl, heteroaryl, carboxy ester, carboxamide, amino,N-acylamino, alkoxy, hydroxy, mercapto, phosphono, sulphono). R⁹ mayalso be an aryl or heteroaryl group that is optionally substituted(alkyl, aryl, heteroaryl, carboxy ester, carboxamide, amino,N-acylamino, alkoxy, hydroxy, mercapto, phosphono).

[0162] (h). Alternatively, the library is assembled with the alkenegroup as the blocked linkage functional group (BLFG). These CTBF's areprepared by condensation of aldehydes or ketone LFG's with phosphorousslides (Maryanoff et al. Chemical Reviews, 89, 863-927 (1989). R⁸ and R⁹may be H or a straight chain or branched alkyl group of length 2 to 10,which may incorporate from 1 to 10 heteroatoms (N, O, P, S) within thechain. R⁸ and R⁹ may also be appended with up to five R¹⁰ groups (R¹⁰ isalkyl, aryl, heteroaryl, carboxy ester, carboxamide, amino, N-acylamino,alkoxy, hydroxy, mercapto, phosphono, sulphono). R⁸ and R⁹ may also bean aryl or heteroaryl group that is optionally substituted (alkyl, aryl,heteroaryl, carboxy ester, carboxamide, amino N-acylamino, alkoxy,hydroxy, mercapto, phosphono, sulphono). R⁸ and R⁹ may also be halogensand heteroatoms.

[0163] (i). Alternatively, carbanions, usually stabilized carbanions,may also be added into the carbonyl. Either the alcohol product A isobtained, or the hydroxyl group is eliminated to provide an alkene B.Numerous methods are available for performing this transformation (Marchet al., supra, 937-950).). R⁸ and R⁹ may be H or a straight chain orbranched alkyl group of length 2 to 10, which may incorporate from 1 to10 heteroatoms (N, O, P, S) within the chain. R⁸ and R⁹ may also beappended with up to five R¹⁰ groups (R¹⁰ is alkyl, aryl, heteroaryl,carboxy ester, carboxamide, amino, N-acylamino, alkoxy, hydroxy,mercapto, phosphono, sulphono). R⁸ and R⁹ may also be an aryl orheteroaryl group that is optionally substituted (alkyl, aryl,heteroaryl, carboxy ester, carboxamide, amino N-acylamino, alkoxy,hydroxy, mercapto, phosphono, sulphono). R⁸ and R⁹ may also be halogensand heteroatoms.

[0164] 2. Primary and Secondary Amines as the Linkage Functional Groupsand the Corresponding Blocked Linkage Functional Groups.

[0165] (a). The linkage functional group is the basic nitrogen ofprimary or secondary amines. These CTBF's may be available commercially,or may be prepared by a variety of known methods to those practiced inthe art. CTBF's that have a primary amine LFG's are represented asfollows.

[0166] CTBF's that have a secondary amine LFG's are represented with twodifferent parts (A and B) of the CTBF attached to the amine group asshown below.

[0167] (b). Alternatively, the library may be assembled as secondary ortertiary amine BLFGs.

[0168] (i). CTBF's with secondary or tertiary amine BLFG's may beprepared by reductive amination of primary amine or secondary amineLFG's, respectively, with aldehydes and ketones. A large number ofreducing agents could be employed that are known to those practiced inthe art (March, et al. supra, pp. 898-900). R⁸ or R⁹ may be H, or astraight chain or branched alkyl group of length 1 to 10, which mayincorporate from 1 to 10 heteroatoms (N, O, P, S) within the chain. R⁸or R⁹ may also be appended with up to five R¹⁰ groups (R¹⁰ is alkyl,aryl, heteroaryl, carboxy ester, carboxamide, amino, N-acylamino,alkoxy, hydroxy, mercapto, phosphono, sulphono). R⁸ or R⁹ may also be anaryl or heteroaryl group that is optionally substituted-(alkyl, aryl,heteroaryl, carboxy ester, carboxamide, amino, N-acylamino, alkoxy,hydroxy, mercapto, phosphono). Numerous reductive amination methods maybe used that are known to those practiced in the art.

[0169] (ii). Alternatively, CTBF's with secondary or tertiary amineBLFG's may be prepared by reaction of primary or secondary amine LFG's,respectively, with an aryl, heteroaryl or alkyl group substituted with aleaving group X, where X may be a halide or a sulfonate group (OSO₂Rwhere R is substituted or unsubstituted alkyl or aryl, e.g. CH₃, CF₃,phenyl-CH₃ and phenyl-NO₂). The halide could be attached to aromatic orheteroaromatic functionality on R⁸, or it could be attached to analiphatic group on R⁸. When X is substituted upon aromatic andheteroaromatic functionality, an S_(N)Ar reaction or apalladium-mediated (or related transtion metal mediated) amine couplingreaction would be performed [e.g, March, et al. supra, pp. 656-657;Wagaw et al., J. Am. Chem. Soc., 119: 8451-8458 (1997) and referencestherein; and Ahman et al. Tetrahedron Letters, 38: 6363-6366 (1997)].Where X is substituted upon alkyl functionality, an S_(N)2 or S_(N)1reaction would be performed (March, et al., supra, pp. 411-413). R⁸ maybe H, or a straight chain or branched alkyl group of length 1 to 10,which may incorporate from 1 to 10 heteroatoms (N, O, P, S) within thechain. R⁸ may also be appended with up to five R⁹ groups (R⁹ is alkyl,aryl, heteroaryl, carboxy ester, carboxamide, amino, N-acylamino,alkoxy, hydroxy, mercapto, phosphono, sulphono). R⁸ may also be an arylor heteroaryl group that is optionally substituted (alkyl, aryl,heteroaryl, carboxy ester, carboxamide, amino, N-acylamino, alkoxy,hydroxy, mercapto, phosphono).

[0170] (iii). Alternatively, CTBF's with secondary or tertiary amineBLFG's may be prepared by reaction of primary or secondary amine LFG's,respectively, with a substituted epoxide (March, et al., supra, p. 416).Many epoxides are available commercially. Alternatively they can beprepared by a number of known methods to those practiced-in the art,most preferably by epoxidation of an alkene. R⁸ to R¹¹ may be H, or astraight chain or branched alkyl group of length 1 to 10, which mayincorporate from 1 to 10 heteroatoms (N, O, P, S) within the chain. R⁸to R¹¹ may also be appended with up to five R¹² groups (R¹² is alkyl,aryl, halide, heteroaryl, carboxy ester, carboxamide, amino,N-acylamino, alkoxy, hydroxy, mercapto, phosphono, sulphono). R⁸ to R¹¹may also be an aryl or heteroaryl group that is optionally substituted(alkyl, aryl, halide, heteroaryl, carboxy ester, carboxamide, amino,N-acylamino, alkoxy, hydroxy, mercapto, phosphono).

[0171] (c). Alternatively, the library may be assembled as amide orthioamide BLFG's. These CTBF's may be prepared by coupling primary andsecondary amine LFG's with carboxylic acids (X═H), carboxylic acidderivatives (X═OR, SR, halide), or the corressponding thiocarboxylicacid derivatives (X═OR, SR, halide). Numerous methods are also availablefor coupling carboxylic acids and carboxylic acid derivatives withamines and are known to those practiced in the art [e.g., March, et al.,supra, pp. 417-425; and Fields et al. Int. J. Peptide Protein Res.35:181-187 (1990)]. R⁸ may be H, or a straight chain or branched alkylgroup of length 1 to 10, which may incorporate from 1 to 10 heteroatoms(N, O, P, S) within the chain. R⁸ may also be appended with up to fiveR⁹ groups (R⁹ is halide, alkyl, aryl, heteroaryl, carboxy ester,carboxamide, amino, N-acylamino, alkoxy, hydroxy, mercapto, phosphono,sulphono). R⁸ may also be an aryl or heteroaryl group that is optionallysubstituted (halide, alkyl, aryl, halide, heteroaryl, carboxy ester,carboxamide, amino, N-acylamino, alkoxy, hydroxy, mercapto, phosphono).

[0172] (d). Alternatively, the library may be assembled as urea orthiourea BLFG's.

[0173] (i). These CTBF's may be prepared by condensation of primary orsecondary amine LFGs and isocyanates or isothiocyanates. The directcoupling of isocyanates and isothiocyanates with amines is known tothose practiced in the art (March, et al., supra, p. 903). R⁸ may be H,or a straight chain or branched alkyl group of length 1 to 10, which mayincorporate from 1 to 10 heteroatoms (N, O, P, S) within the chain. R⁸may also be appended with up to five R⁹ groups (R⁹ is alkyl, aryl,heteroaryl, carboxy ester, carboxamide, amino, N-acylamino, alkoxy,hydroxy, mercapto, phosphono, sulphono). R⁸ may also be an aryl orheteroaryl group that is optionally substituted (alkyl, aryl,heteroaryl, carboxy ester, carboxamide, amino N-acylamino, alkoxy,hydroxy, mercapto, phosphono).

[0174] (ii). Alternatively, these CTBF's may be prepared by a two stepprocess. In the first step, the primary or secondary amine LFG's isconverted to carbamate (thiocarbamates) or related derivatives where Xand Y are alkoxy groups, mercaptyl groups, halides, or other suitableleaving groups. In the second step a primary or secondary amine is added(e.g., Hutchins, Tetrahedron Letters, 35: 4055-4058, (1994) andreferences therein). R⁸ and R⁹ may be H, or a straight chain or branchedalkyl groups of length 1 to 10, which may incorporate from 1 to 10heteroatoms (N, O, P, S) within the chain. R⁸ and R⁹ may also beappended with up to five R¹⁰ groups (R¹⁰ is alkyl, aryl, heteroaryl,carboxy ester, carboxamide, amino, N-acylamino, alkoxy, hydroxy,mercapto, phosphono, sulphono). R⁸ and R⁹ may also be aryl or heteroarylgroups that is optionally substituted (alkyl, aryl, heteroaryl, carboxyester, carboxamide, amino N-acylamino, alkoxy, hydroxy, mercapto,phosphono).

[0175] (iii). These CTBF's may also be prepared by condensation ofprimary or secondary amine LFG's and carbamates, thiocarbamates, orrelated derivatives where X is an alkoxy group, a mercaptyl group, or ahalide (e.g., Hutchins, Tetrahedron Letters, 35: 4055-4058, (1994) andreferences therein). R⁸ and R⁹ may be H, or a straight chain or branchedalkyl group of length 1 to 10, which may incorporate from 1 to 10heteroatoms (N, O, P, S) within the chain. R⁸ and R⁹ may also beappended with up to five R¹⁰ groups (R¹⁰ is alkyl, aryl, heteroaryl,carboxy ester, carboxamide, amino, N-acylamino, alkoxy, hydroxy,mercapto, phosphono, sulphono). R⁸ and R⁹ may also be aryl or heteroarylgroups that are optionally substituted (alkyl, aryl, heteroaryl, carboxyester, carboxamide, amino N-acylamino, alkoxy, hydroxy, mercapto,phosphono).

[0176] (d). Alternatively, the library may be assembled as sulfonamideBLFG's. These CTBF's may be prepared by coupling primary and secondaryamine LFG's with sulfonic acids (X═H) or sulfonic acid derivatives(X═OR, SR, halide). Numerous methods are available for coupling sulfonicacids and sulfonic acid derivatives with amines and are known to thosepracticed in the art (e.g., March, et al., supra, p. 499, Greene, et al.Protective Groups in Organic Synthesis, John Wiley & Sons, New York,2^(nd) edition, 1991, pp. 379-385). R⁸ may be H, or a straight chain orbranched alkyl group of length 1 to 10, which may incorporate from 1 to10 heteroatoms (N, O, P, S) within the chain. R⁸ may also be appendedwith up to five R⁹ groups (R⁹ is halide, alkyl, aryl, heteroaryl,carboxy ester, carboxamide, amino, N-acylamino, alkoxy, hydroxy,mercapto, phosphono, sulphono). R⁸ may also be an aryl or heteroarylgroup that is optionally substituted (halide, alkyl, aryl, halide,heteroaryl, carboxy ester, carboxamide, amino, N-acylamino, alkoxy,hydroxy, mercapto, phosphono).

[0177] (e). Alternatively, the library may be assembled as carbamate,thiocarbamate or related BLFG's. These CTBF's may be prepared bycondensation of primary and secondary amine pharmacophores with carbonylderivatives (X=halide, OR, SR; Y═S,O; Z═S,O). Numerous methods are knownto those practiced in the art (e.g., March, et al., supra, p. 418; andGreene, et al., supra, pp. 315-348). R⁸ may be H, or a straight chain orbranched alkyl group of length 1 to 10, which may incorporate from 1 to10 heteroatoms (N, O, P, S) within the chain. R⁸ may also be appendedwith up to five R⁹ groups (R⁹ is alkyl, aryl, heteroaryl, carboxy ester,carboxamide, amino, N-acylamino, alkoxy, hydroxy, mercapto, phosphono,sulphono). R⁸ may also be an aryl or heteroaryl group that is optionallysubstituted (alkyl, aryl, heteroaryl, carboxy ester, carboxamide, aminoN-acylamino, alkoxy, hydroxy, mercapto, phosphono, sulfono).

[0178] (f). Alternatively, the library may be assembled as guanidineBLFGs. These CTBFs may be prepared by condensation of primary andsecondary amine pharmacophores with carbonyl derivatives (X=halide,OSO₂R, OR, SR) [Roskamp, et al. Tetrahedron, 53:6697-6705 (1997)]. R⁸may be H, or a straight chain or branched alkyl group of length 1 to 10,which may incorporate from 1 to 10 heteroatoms (N, O, P, S) within thechain. R⁸ may also be appended with up to five R⁹ groups (R⁹ is alkyl,aryl, heteroaryl, carboxy ester, carboxamide, amino, N-acylamino,alkoxy, hydroxy, mercapto, phosphono, sulphono). R⁸ may also be adn arylor heteroaryl group that is optionally substituted (alkyl, aryl,heteroaryl, carboxy ester, carboxamide, amino N-acylamino, alkoxy,hydroxy, mercapto, phosphono, sulfono).

[0179] 3. Epoxides as the Linkage Functional Groups and theCorresponding Blocked Linkage Functional Groups.

[0180] (a). The linkage functional group is the epoxide group. TheseCTBFs may be available commercially, or may be prepared by a variety ofknown methods to those practiced in the art.

[0181] Epoxide CTBFs are represented with four parts (A through D) ofthe CTBF attached to the epoxide group as shown below. Each fragment maybe H, or functionality whereby a carbon atom is directly attached to theepoxide functionality.

[0182] (b). Alternatively, the library may be assembled as 1,2-aminoalcohol BLFGs. These CTBFs may be prepared by coupling epoxide LFGs withprimary or secondary amines employing known methods to those practicedin the art (March, et al., supra, p. 416). R⁸ or R⁹ may be H, or astraight chain or branched alkyl group of length 1 to 10, which mayincorporate from 1 to 10 heteroatoms (N, O, P, S) within the chain. R⁸or R⁹ may also be appended with up to five R¹⁰ groups (R¹⁰ is alkyl,aryl, heteroaryl, carboxy ester, carboxamide, amino, N-acylamino,alkoxy, hydroxy, mercapto, phosphono, sulphono). R⁸ or R⁹ may also be anaryl or heteroaryl group that is optionally substituted (alkyl, aryl,heteroaryl, carboxy ester, carboxamide, amino, N-acylamino, alkoxy,hydroxy, mercapto, phosphono).

[0183] (c). Alternatively, the library may be assembled as, 2-hydroxythioether BLFGs. These CTBFs may be prepared by coupling epoxide LFGswith thiols employing known methods to those practiced in the art(Wardell, in Patai The Chemistry of the Thiol Group, pt. 1; Wiley, NewYork, 1974, pp. 246-251). R⁸ may be H, or a straight chain or branchedalkyl group of length 1 to 10, which may incorporate from f to 10heteroatoms (N, O, P, S) within the chain. R⁸ may also be appended withup to five R⁹ groups (R⁹ is alkyl, aryl, heteroaryl, carboxy ester,carboxamide, amino, N-acylamino, alkoxy, hydroxy, mercapto, phosphono,sulphono). R⁸ may also be an aryl or heteroaryl group that is optionallysubstituted (alkyl, aryl, heteroaryl, carboxy ester, carboxamide, amino,N-acylamino, alkoxy, hydroxy, mercapto, phosphono). The 2-hydroxythioether BLFGs may also be readily oxidized to more water soluble2-hydroxy sulfoxide or sulfone BLFGs (March, et al., supra, pp.1201-1203).

[0184] (d). Alternatively, the library may be assembled as,2-hydroxyether BLFGs. These CTBFs may be prepared by coupling epoxide LFGs withalcohols employing known methods to those practiced in the art (March,et al., supra, p. 391). R8 may be H, or a straight chain or branchedalkyl group of length 1 to 10, which may incorporate from 1 to 10heteroatoms (N, O, P, S) within the chain. R⁸ may also be appended withup to five R⁹ groups (R⁹ is alkyl, aryl, heteroaryl, carboxy ester,carboxamide, amino, N-acylamino, alkoxy, hydroxy, mercapto, phosphono,sulphono). R⁸ may also be an aryl or heteroaryl group that is optionallysubstituted (alkyl, aryl, heteroaryl, carboxy ester, carboxamide, amino,N-acylamino, alkoxy, hydroxy, mercapto, phosphono).

[0185] 4. Carboxylic Acids as the Linkage Functional Groups and theCorresponding Blocked Linkage Functional Groups

[0186] (a). The linkage functional group is the carboxylic acid. TheseCTBFs may be available commercially, or may be prepared by a variety ofknown methods to those practiced in the art.

[0187] Carboxylic acid CTBFs are represented as follows.

[0188] (b). Alternatively, the library may be assembled as amide BLFGs.These CTBFs may be prepared by coupling carboxylic acid LFGs (X═OH), orderivatives of carboxylic acid LFGs (X═OR, SR, halide) with primary orsecondary amines. Numerous known methods are available for couplingcarboxylic acids and carboxylic acid derivatives with amines to thosepracticed in the art [e.g., March, et al., supra, pp. 417-425; andFields et al. Int. J. Peptide Protein Res. 35:181-187 (1990)]. R⁸ and R⁹may be H, or a straight chain or branched alkyl group of length 1 to 10,which may incorporate from 1 to 10 heteroatoms (N, O, P, S) within thechain. R⁸ and R⁹ may also be appended with up to five R¹⁰ groups (R¹⁰ isalkyl, aryl, heteroaryl, carboxy ester, carboxamide, amino, N-acylamino,alkoxy, hydroxy, mercapto, phosphono, sulphono). R⁸ and R⁹ may also bean aryl or heteroaryl group that is optionally substituted (alkyl, aryl,halide, heteroaryl, carboxy ester, carboxamide, amino, N-acylamino,alkoxy, hydroxy, mercapto, phosphono).

[0189] (c). Alternatively, amine BLFGs could be prepared by reduction ofamide BLFGs prepared as described above in step 4b from thecorresponding carboxylic acid LFGs. A number of reducing agents could beemployed that are known to those practiced in the art (e.g., March, etal., supra, pp. 1212-1213). R⁸ or R⁹ may be H, or a straight chain orbranched alkyl group of length 1 to 10, which may incorporate from 1 to10 heteroatoms (N, O, P, S) within the chain. R⁸ or R⁹ may also beappended with up to five R¹⁰ groups (R¹⁰ is alkyl, aryl, heteroaryl,carboxy ester, carboxamide, amino, N-acylamino, alkoxy, hydroxy,mercapto, phosphono, sulphono). R⁸ or R⁹ may also be an aryl orheteroaryl group that is optionally substituted (alkyl, aryl,heteroaryl, carboxy ester, carboxamide, amino, N-acylamino, alkoxy,hydroxy, mercapto, phosphono).

[0190] (d). Alternatively, the library may be assembled as ester BLFGs.

[0191] (i). These CTBFs may be prepared by coupling carboxylic acid LFGs(X═OH), or derivatives of carboxylic acid LFGs (X═OR, SR, halide) withalcohols. Carboxylic acids and carboxylic acid derivatives may becoupled with alcohols employing numerous known methods to thosepracticed in the art [e.g., (March, et al., supra, pp. 392-398) and(e.g., Greene, et al., supra, pp. 227-228), and (Hughes et al.(Paquette,Series Editor in Chief), Organic Reactions, John Wiley & Sons, New York,1992, vol. 42, pp. 343-347)]. R⁸ may be H, or a straight chain orbranched alkyl group of length 1 to 10, which may incorporate from 1 to10 heteroatoms (N, O, P, S) within the chain. R⁸ may also be appendedwith up to five R⁹ groups (R⁹ is alkyl, aryl, heteroaryl, carboxy ester,carboxamide, amino, N-acylamino, alkoxy, hydroxy, mercapto, phosphono,sulphono).% R⁸ may also be an aryl or heteroaryl group that isoptionally substituted (alkyl, aryl, halide, heteroaryl, carboxy ester,carboxamide, amino, N-acylamino, alkoxy, hydroxy, mercapto, phosphono).

[0192] (ii). These CTBFs may be prepared by reacting carboxylic acidLFGs with an aryl, heteroaryl or alkyl group substituted with a leavinggroup X, where X may be a halide or a sulfonate group (OSO₂R where R issubstituted or unsubstituted alkyl or aryl, e.g. CH₃, CF₃, phenyl-CH₃and phenyl-NO₂). The halide could be attached to aromatic orheteroaromatic functionality on R⁸, or it could be attached to analiphatic group on R⁸. When X is substituted upon aromatic andheteroaromatic functionality, an S_(N)Ar reaction or apalladium-mediated, copper-mediated or related transtion metal mediatedcoupling reaction would be performed. Where X is substituted upon alkylfunctionality, an S_(N)2 or S_(N)1 reaction would be performed. Methodsfor these transformations are known to those practiced in the art [e.g.,(March, et al., supra, pp. 398-399) and (e.g., Greene, et al., supra,pp. 228-229)]. R⁸ may be H, or a straight chain or branched alkyl groupof length 1 to 10, which may incorporate from 1 to 10 heteroatoms (N, O,P, S) within the chain. R⁸ may also be appended with up to five R⁹groups (R⁹ is alkyl, aryl, heteroaryl, carboxy ester, carboxamide,amino, N-acylamino, alkoxy, hydroxy, mercapto, phosphono, sulphono). R⁸may also be an aryl or heteroaryl group that is optionally substituted(alkyl, aryl, heteroaryl, carboxy ester, carboxamide, amino,N-acylamino, alkoxy, hydroxy, mercapto, phosphono).

[0193] (e). Alternatively the library may be assembled as thioesterBLFGs.

[0194] (i). These CTBFs may be prepared by condensation of carboxylicacid LFGs (X═OH), or carboxylic acid derivatives (X═OR, SR, halide) andthiols. Carboxylic acids and carboxylic acid derivatives may be coupledwith thiols employing known methods to those practiced in the art (e.g.,March, et al., supra, p. 409). R⁸ may be H, or a straight chain orbranched alkyl group of length 1 to 10, which may incorporate from 1 to10 heteroatoms (N, O, P, S) within the chain. R⁸ may also be appendedwith up to five R⁹ groups (R⁹ is alkyl, aryl, heteroaryl, carboxy ester,carboxamide, amino, N-acylamino, alkoxy, hydroxy, mercapto, phosphono,sulphono). R⁸ may also be an aryl or heteroaryl group that is optionallysubstituted (alkyl, aryl, halide, heteroaryl, carboxy ester,carboxamide, amino, N-acylamino, alkoxy, hydroxy, mercapto, phosphono).

[0195] 5. Sulfonic Acids as the Linkage Functional Groups and theCorresponding Blocked Linkage Functional Groups

[0196] (a). The linkage functional group is the sulfonic acid. TheseCTBFs may be available commercially, or may be prepared by a variety ofknown methods to those practiced in the art. Sulfonic acid CTBFs arerepresented as follows.

[0197] (b). Alternatively, the library may be assembled as sulfonamideBLFGs. These CTBFs may be prepared by reacting sulfonic acid LFGs (X═OH)or derivatives of sulfonic acids (X=halide, alkoxyl, mercaptyl) withamines (e.g., March, et al., supra, p. 499, Greene, et al. supra, pp.379-385). R⁸ and R⁹ may be H, or a straight chain or branched alkylgroup of length 1 to 10, which may incorporate from 1 to 10 heteroatoms(N, O, P, S) within the chain. R⁸ and R⁹ may also be appended with up tofive R¹⁰ groups (R¹⁰ is alkyl, aryl, heteroaryl, carboxy ester,carboxamide, amino, N-acylamino, alkoxy, hydroxy, mercapto, phosphono,sulphono). R⁸ and R⁹ may also be an aryl or heteroaryl group that isoptionally substituted (alkyl, aryl, heteroaryl, carboxy ester,carboxamide, amino N-acylamino, alkoxy, hydroxy, mercapto, phosphono).

[0198] 6. The Hydroxyl Group as the Linkage Functional Group and theCorresponding Blocked Linkage Functional Groups

[0199] (a). The linkage functional group is the hydroxyl group.

[0200] These CTBFs may be available commercially, or may be prepared bya variety of known methods to those practiced in the art.

[0201] Alcohol CTBFs are represented as follows.

[0202] (b). Alternatively the library may be assembled as ether BLFGs.

[0203] (i). The ether may be prepared by reaction of an alcohol LFG withan aryl, heteroaryl or alkyl group substituted with a leaving group X,where X may be a halide or a sulfonate group (OSO₂R where R issubstituted or unsubstituted alkyl or aryl, e.g. CH₃, CF₃, phenyl-CH₃and phenyl-NO₂). The halide could be attached to aromatic orheteroaromatic functionality on R⁸, or it could be attached to analiphatic group on R⁸. When X is substituted upon aromatic andheteroaromatic functionality, an S_(N)Ar reaction or apalladium-mediated, copper-mediated or related transtion-metal mediatedcoupling reaction would be performed [(e.g., March, et al., supra, pp.654-655) and Hartwig et al., supra, pp. 8005-8008). Where X issubstituted upon alkyl functionality, an S_(N)2 or S_(N)1 reaction wouldbe performed. R⁸ may be H, or a straight chain or branched alkyl groupof length 1 to 10, which may incorporate from 1 to 10 heteroatoms (N, O,P, S) within the chain. R⁸ may also be appended with up to five R⁹groups (R⁹ is alkyl, aryl, heteroaryl, carboxy ester, carboxamide,amino, N-acylamino, alkoxy, hydroxy, mercapto, phosphono, sulphono). R⁸may also be an aryl or heteroaryl group that is optionally substituted(alkyl, aryl, heteroaryl, carboxy ester, carboxamide, amino,N-acylamino, alkoxy, hydroxy, mercapto, phosphono).

[0204] (ii). The ether may be prepared by the Mitsunobu reaction betweenalcohol LFGs and another alcohol where the second alcohol is acidic(pKa≦12), for example, phenols and oximes (Hughes et al.(Paquette,Series Editor in Chief), Organic Reactions, John Wiley & Sons, New York,1992, vol. 42, pp. 335-636). R⁸ may be H, or a straight chain orbranched alkyl group of length 1 to 10, which may incorporate from 1 to10 heteroatoms (N, O, P, S) within the chain. R⁸ may also be appendedwith up to five R⁹ groups (R⁹ is alkyl, aryl, heteroaryl, carboxy ester,carboxamide, amino, N-acylamino, alkoxy, hydroxy, mercapto, phosphono,sulphono). R⁸ may also be an aryl or heteroaryl group that is optionallysubstituted (alkyl, aryl, heteroaryl, carboxy ester, carboxamide, amino,N-acylamino, alkoxy, hydroxy, mercapto, phosphono). The key requirementon R⁸ is that the alcohol has a pKa≦12.

[0205] (iii). The ether may also be prepared by the Mitsunobu reactionbetween alcohol LFGs and another alcohol, where the alcohol LFGs areacidic (pKa<12), for example, phenols or oximes (Hughes et al.(Paquette,Series Editor in Chief), Organic Reactions, John Wiley & Sons, New York,1992, vol. 42, pp. 335-636). R⁸ may be H, or a straight chain orbranched alkyl group of length 1 to 10, which may incorporate from 1 to10 heteroatoms (N, O, P, S) within the chain. R⁸ may also be appendedwith up to five R⁹ groups (R⁹ is alkyl, aryl, heteroaryl, carboxy ester,carboxamide, amino, N-acylamino, alkoxy, hydroxy, mercapto, phosphono,sulphono). The key requirement on the pharmacophore substituted alcoholis that it has a pK_(a)<12.

[0206] (c). Alternatively the library may be assembled as ester (Z=O) orthioester (Z=S) BLFGs. Alcohol LFGs may be coupled with carboxylic acids(Z=0, X═OH), carboxylic acid derivatives (Z=O, X═OR, SR, halide), or thecorresponding thio-substituted derivatives (Z=S, X═OH, OR, SR halide).Carboxylic acids and carboxylic acid derivatives may be coupled withalcohols employing a variety of known methotos-to those practiced in theart [e.g., (March, et al., supra, pp. 392-398) and (e.g., Greene, etal., supra, pp. 227-228), and (Hughes et al.(Paquette, Series Editor inChief), Organic Reactions, John Wiley & Sons, New York, 1992, vol. 42,pp. 343-347)]. R⁸ may be H, or a straight chain or branched alkyl groupof length 1 to 10, which may incorporate from 1 to 10 heteroatoms (N, O,P, S) within the chain. R⁸ may also be appended with up to five R⁹groups (R⁹ is halide, alkyl, aryl, heteroaryl, carboxy ester,carboxamide, amino, N-acylamino, alkoxy, hydroxy, mercapto, phosphono,sulphono). R⁸ may also be an aryl or heteroaryl group that is optionallysubstituted (halide, alkyl, aryl, halide, heteroaryl, carboxy ester,carboxamide, amino, N-acylamino, alkoxy, hydroxy, mercapto, phosphono).

[0207] (d). Alternatively the library may be assembled from thioetherBLFGs. The thioethers may be prepared by the Mitsunobu reaction betweenalcohol LFGs and a thiol (Hughes et al.(Paquette, Series Editor inChief), Organic Reactions, John Wiley & Sons, New York, 1992, vol. 42,pp. 365-366, 381-382). R⁸ may be H, or a straight chain or branchedalkyl group of length 1 to 10, which may incorporate from 1 to 10heteroatoms (N, O, P, S) within the chain. R⁸ may also be appended withup to five R⁹ groups (R⁹ is alkyl, aryl, heteroaryl, carboxy ester,carboxamide, amino, N-acylamino, alkoxy, hydroxy, mercapto, phosphono,sulphono). R⁸ may also be an aryl or heteroaryl group that is optionallysubstituted (alkyl, aryl, heteroaryl, carboxy ester, carboxamide, amino,N-acylamino, alkoxy, hydroxy, mercapto, phosphono). The thioether BLFGsmay also be readily oxidized to more water soluble sulfoxide or sulfoneBLFGs.

[0208] (e). Alternatively the library may be assembled as carbamate(Z=0) or thiocarbamate (Z=S) BLFGs.

[0209] (i). These CTBFs may be prepared by condensation of alcohol LFGsand isocyanates or isothiocyanates. The direct coupling of isocyanatesand isothiocyanates with alcohols is straighforward and obvious to thosepracticed in the art (March, et al., supra, pp. 891-892). R⁸ may be H,or a straight chain or branched alkyl group of length 1 to 10, which mayincorporate from 1 to 10 heteroatoms (N, O, P, S) within the chain. R⁸may also be appended with up to five R⁹ groups (R⁹ is alkyl, aryl,heteroaryl, carboxy ester, carboxamide, amino, N-acylamino, alkoxy,hydroxy, mercapto, phosphono, sulphono). R⁸ may also be an aryl orheteroaryl group that is optionally substituted (alkyl, aryl,heteroaryl, carboxy ester, carboxamide, amino N-acylamino, alkoxy,hydroxy, mercapto, phosphono).

[0210] (ii). These CTBFs may be prepared by condensation of alcohol LFGsand carbamates, thiocarbamates, or related derivatives where X is analkoxy group, a mercaptyl group, or a halide. R⁸ may be H, or a straightchain or branched alkyl group of length 1 to 10, which may incorporatefrom 1 to 10 heteroatoms (N, O, P, S) within the chain. R⁸ may also beappended with up to five R⁹ groups (R⁹ is alkyl, aryl, heteroaryl,carboxy ester, carboxamide, amino, N-acylamino, alkoxy, hydroxy,mercapto, phosphono, sulphono). R⁸ may also be an aryl or heteroarylgroup that is optionally substituted (alkyl, aryl, heteroaryl, carboxyester, carboxamide, amino N-acylamino, alkoxy, hydroxy, mercapto,phosphono).

[0211] (iii). These CTBFs may be prepared in a two step process (e.g.,March, et al., supra, p. 418; and Greene, et al., supra, pp. 315-348).In the first step, the alcohol LFGs are converted to carbonate (Z=O) orthiocarbonate (Z=S) or related derivatives where X and Y are alkoxygroups, mercaptyl groups, halides, or other suitable leaving groups. Inthe second step an amine is added to, displace the leaving group Y. R⁸and R⁹ may be H, or a straight chain or branched alkyl groups of length1 to 10, which may incorporate from 1 to 10 heteroatoms (N, O, P, S)within the chain. R⁸ and R⁹ may also be appended with up to five R¹⁰groups (R¹⁰ is alkyl, aryl, heteroaryl, carboxy ester, carboxamide,amino, N-acylamino, alkoxy, hydroxy, mercapto, phosphono, sulphono). R⁸and R⁹ may also be an aryl or heteroaryl group that is optionallysubstituted (alkyl, aryl, heteroaryl, carboxy ester, carboxamide, aminoN-acylamino, alkoxy, hydroxy, mercapto, phosphono).

[0212] (f). Alternatively the library may be assembled with thioesterBLFGs. The thioesters may be prepared by the Mitsunobu reaction betweenalcohol LFGs and a thiol acid (Hughes et al.(Paquette, Series Editor inChief), Organic Reactions, John Wiley & Sons, New York, 1992, vol. 42,pp. 343-347). R8 may be H, or a straight chain or branched alkyl groupof length 1 to 10, which may incorporate from 1 to 10 heteroatoms (N, O,P, S) within the chain. R8 may also be appended with up to five R⁹groups (R⁹ is alkyl, aryl, heteroaryl, carboxy ester, carboxamide,amino, N-acylamino, alkoxy, hydroxy, mercapto, phosphono, sulphono). R⁸may also be an aryl or heteroaryl group that is optionally substituted(alkyl, aryl, heteroaryl, carboxy ester, carboxamide, amino,N-acylamino, alkoxy, hydroxy, mercapto, phosphono).

[0213] (g). Alternatively the library may be assembled with amide,thioamide, urea, thiourea, sulfonamide, carbamate, thiocarbamate (Z=SO₂,CO, CS, CO₂, COS, CSO, CONR¹¹, CSNR¹¹.) BLFGs. Substitution of thehydroxyl LFGs are accomplished using the Mitsunobu reaction [e.g.,(Hughes et al.(Paquette, Series Editor in Chief), Organic Reactions,John Wiley & Sons, New York, 1992, vol. 42, pp. 335-636) and (Fukuyama,et al., Tetrahedron Letters, 38: 5831-5834 (1997) and referencestherein)]. An aliphatic hydroxyl group is prefered. R⁸ and R⁹ may be H,or a straight chain or branched alkyl group of length 1 to 10, which mayincorporate from 1 to 10 heteroatoms (N, O, P, S) within the chain. R⁸and R⁹ may also be appended with up to five R¹⁰ groups (R¹⁰ is alkyl,aryl, halide, heteroaryl, carboxy ester, carboxamide, amino,N-acylamino, alkoxy, hydroxy, mercapto, phosphono, sulphono). R⁸ and R⁹may also be an aryl or heteroaryl group that is optionally substituted(alkyl, aryl, halide, heteroaryl, carboxy ester, carboxamide, amino,N-acylamino, alkoxy, hydroxy, mercapto, phosphono).

[0214] (h). Alternatively, the library man be assembled with amineBLFGs. The amine BLFGs will be prepared from the BLFGs described in 6g.The acyl or sulfonyl functionality may be removed by methods known tothose practiced in the art (such as, acidic or basic hydrolysis, ordissolving metal reactions for sulfonamides Greene et al., supra,349-357 and 379-385). Milder conditions may be applied to morespecialized groups, e.g., trifluoroacetamides may be cleaved by mildbasic hydrolysis (Greene et al., supra, 353-354), and nitrosubstitutedbenzenesulfonamides may be cleaved by thiolate addition) to providesecondary amines (Fukuyama, et al., Tetrahedron Letters, 38: 5831-5834(1997) and references therein). Alternatively for some derivatives,reduction will provide tertiary amines (e.g., March, et al., supra, pp.1212-1213).

[0215] 7. The Thiol Group as the Linkage Functional Group and theCorresponding Blocked Linkage Functional Groups

[0216] (a). The linkage functional group is the thiol group. These CTBFsmay be available commercially, or may be prepared by a variety of knownmethods to those practiced in the art.

[0217] Thiol CTBFs are represented as follows.

[0218] (b). Alternatively the library may be assembled as thioetherBLFGs.

[0219] (i). The thioether may be prepared by reaction of a thiol LFGwith an aryl, heteroaryl or alkyl group substituted with a leaving groupX, where X may be a halide or a sulfonate group (OSO₂R where R issubstituted or unsubstituted alkyl or aryl, e.g. CH₃, CF₃, phenyl-CH₃and phenyl-NO₂). The halide could be attached to aromatic orheteroaromatic functionality on R⁸, or it could be attached to analiphatic group on R⁸. When X is substituted upon aromatic andheteroaromatic functionality, an S_(N)Ar reaction or apalladium-mediated, copper-mediated or related transtion metal mediatedcoupling reaction would be performed. Where X is substituted upon alkylfunctionality, an S_(N)2 or S_(N)1 reaction would be performed. Thesemethods are known to those practiced in the art [(March et al., supra,pp. 407-409) and (Peach in Patai The Chemistry of the Thiol Group, pt.1, John Wiley & Sons, New York, 1974, pp. 721-735)]. R⁸ may be H, or astraight chain or branched alkyl group of length 1 to 10, which mayincorporate from 1 to 10 heteroatoms (N, O, P, S) within the chain. R⁸may also be appended with up to five R⁹ groups (R⁹ is alkyl, aryl,heteroaryl, carboxy ester, carboxamide, amino, N-acylamino, alkoxy,hydroxy, mercapto, phosphono, sulphono). R⁸ may also be an aryl orheteroaryl group that is optionally substituted (alkyl, aryl,heteroaryl, carboxy ester, carboxamide, amino, N-acylamino, alkoxy,hydroxy, mercapto, phosphono). The thioether BLFGs may also b oxidizedto more water soluble sulfoxide and sulfone BLFGs (March, et al., supra,pp. 1201-1203).

[0220] (ii). The thioether may also be prepared by the Mitsunobureaction between alcohol LFGs and a thiol (e.g., (Hughes et al.(Paquette, Series Editor in Chief), Organic Reactions, John Wiley &Sons, New York, 1992, vol. 42, pp. 335-636). R⁸ may be H, or a straightchain or branched alkyl group of length 1 to 10, which may incorporatefrom 1 to 10 heteroatoms (N, O, P, S) within the chain. R⁸ may also beappended with up to five R⁹ groups (R⁹ is alkyl, aryl, heteroaryl,carboxy ester, carboxamide, amino, N-acylamino, alkoxy, mercapto,phosphono, sulphono). The thioether BLFGs may also b oxidized to morewater soluble sulfoxide and sulfone BLFGs (March, et al., supra, pp.1201-1203).

[0221] (c). Alternatively the library may be assembled with BLFGswhereby the thiol LFGs are acylated (March et al., supra, p. 409). ThiolLFGs may be coupled with carboxylic acids (Z=O, X═H), carboxylic acidderivatives (Z=O, X═OR, SR, halide), or the correspondingthio-substituted derivatives (Z=S, X═H, OR, SR halide). Carboxylic acidsand carboxylic acid derivatives may be coupled with thiols employingknown methods to those practiced in the art. R⁸ may be H, or a straightchain or branched alkyl group of length 1 to 10, which may incorporatefrom 1 to 10 heteroatoms (N, O, P, S) within the chain. R⁸ may also beappended with up to five R⁹ groups (R⁹ is halide, alkyl, aryl,heteroaryl, carboxy ester, carboxamide, amino, N-acylamino, alkoxy,hydroxy, mercapto, phosphono, sulphono). R⁸ may also be an aryl orheteroaryl group that is optionally substituted (halide, alkyl, aryl,halide, heteroaryl, carboxy ester, carboxamide, amino, N-acylamino,alkoxy, hydroxy, mercapto, phosphono).

[0222] (d). Alternatively the library may be assembled as thiocarbamate(Z=O) or thiocarbamate (Z=S) BLFGs.

[0223] (i). These CTBFs may be prepared by condensation of thiol LFGsand isocyanates or isothiocyanates. The direct coupling of isocyanatesand isothiocyanates with thiols is straighforward and obvious to thosepracticed in the art (Greene et al. supra, p. 301). R⁸ may be H, or astraight chain or branched alkyl group of length 1 to 10, which mayincorporate from 1 to 10 heteroatoms (N, O, P, S) within the chain. R⁸may also be appended with up to five R⁹ groups (R⁹ is alkyl, aryl,heteroaryl, carboxy ester, carboxamide, amino, N-acylamino, alkoxy,hydroxy, mercapto, phosphono, sulphono). R⁸ may also be an aryl orheteroaryl group that is optionally substituted (alkyl, aryl,heteroaryl, carboxy ester, carboxamide, amino N-acylamino, alkoxy,hydroxy, mercapto, phosphono).

[0224] (ii). These CTBFs may be prepared by condensation of thiol LFGsand carbamates, thiocarbamates, or related derivatives where X is analkoxy group, a mercaptyl group, or a halide. R⁸ may be H, or a straightchain or branched alkyl group of length 1 to 10, which may incorporatefrom 1 to 10 heteroatoms (N, O, P, S) within the chain. R⁸ may also beappended with up to five R⁹ groups (R⁹ is alkyl, aryl, heteroaryl,carboxy ester, carboxamide, amino, N-acylamino, alkoxy, hydroxy,mercapto, phosphono, sulpheno). R⁸ may also be an aryl or heteroarylgroup that is optionally substituted (alkyl, aryl, heteroaryl, carboxyester, carboxamide, amino N-acylamino, alkoxy, hydroxy, mercapto,phosphono).

[0225] (iii). These CTBFs may be prepared in a two step process. In thefirst step, the thiol LFGs are converted to carbonate (Z=O) orthiocarbonate (Z=S) or related derivatives where X and Y are alkoxygroups, mercaptyl groups, halides, or other suitable leaving groups(Greene et al. supra, pp. 299-301). In the second step an amine is addedto displace the leaving group Y. R⁸ and R⁹ may be H, or a straight chainor branched alkyl groups of length 1 to 10, which may incorporate from 1to 10 heteroatoms (N, O, P, S) within the chain. R⁸ and R⁹ may also beappended with Lip to five R¹⁰ groups (R¹⁰ is alkyl, aryl, heteroaryl,carboxy ester, carboxamide, amino, N-acylamino, alkoxy, hydroxy,mercapto, phosphono, sulphono). R⁸ and R⁹ may also be an aryl orheteroaryl group that is optionally substituted (alkyl, aryl,heteroaryl, carboxy ester, carboxamide, amino N-acylamino, alkoxy,hydroxy, mercapto, phosphono).

[0226] (e). Alternatively the library may be assembled as disulfideBLFGs. The disulfides may be prepared by reacting the thiol LFGs withthiols (X═H) or activated thiols (X=mercaptyl, halide, sulfonyl)employing known methods to those practiced in the art (e.g., Greene etal., supra, pp. 302-303). R⁸ may be H, or a straight chain or branchedalkyl group of length 1 to 10, which may incorporate from 1 to 10heteroatoms (N, O, P, S) within the chain. R⁸ may also be appended withup to fine R⁹ groups (R⁹ is halide, alkyl, aryl, heteroaryl, carboxyester, carboxamide, amino, N-acylamino, alkoxy, hydroxy, mercapto,phosphono, sulphono). R⁸ may also be an aryl or heteroaryl group that isoptionally substituted (halide, alkyl, aryl, halide, heteroaryl, carboxyester, carboxamide, amino, N-acylamino, alkoxy, hydroxy, mercapto,phosphono).

[0227] 8. The Isocyanate or Isothiocyanate Group as the LinkageFunctional Group and the Corresponding Blocked Linkage Functional Groups

[0228] (a). The linkage functional group is the isocyanate (Z=0) orisothiocyanate (Z=S) group. These CTBFs may be available commercially,or may be prepared by a variety of known methods to those practiced inthe art.

[0229] Isocyanate (Z=O) or isothiocyanate (Z=S) CTBFs are represented asfollows.

[0230] (b). Alternatively the library may be assembled with urea orthiourea BLFGS by reaction of the isocyanate (Z=O) or isothiocyanate(Z=S) LFGs with amines (March et al., supra, p. 903). The directcoupling of isocyanates with amines is straighforward and known to thosepracticed in the art. R⁸ and R⁹ may be H, or a straight chain orbranched alkyl groups of length 1 to 10, which may incorporate from 1 to10 hetereatoms (N, O, P, S) within the chain. R⁸ and R⁹ may also beappended with up to five R¹⁰ groups (R¹⁰ is alkyl, aryl, heteroaryl,carboxy ester, carboxamide, amino, N-acylamino, alkoxy, hydroxy,mercapto, phosphono, sulphono). R⁸ and R⁹ may also be an aryl orheteroaryl group that is optionally substituted (alkyl, aryl,heteroaryl, carboxy ester, carboxamide, amino N-acylamino, alkoxy,hydroxy, mercapto, phosphono).

[0231] (c). Alternatively the library may be assembled as BLFGs byreaction of the isocyanate (Z=O) or isothiocyanate (Z=S) LFGs withthiols (Green et al., supra, p. 301). R⁸ may be H, or a straight chainor branched alkyl groups of length 1 to 10, which may incorporate from 1to 10 heteroatoms (N, O, P, S) within the chain. R⁸ may also be appendedwith up to five R⁹ groups (R⁹ is alkyl, aryl, heteroaryl, carboxy ester,carboxamide, amino, N-acylamino, alkoxy, hydroxy, mercapto, phosphono,sulphono). R⁸ may also be an aryl or heteroaryl group that is optionallysubstituted (alkyl, aryl, heteroaryl, carboxy ester, carboxamide, aminoN-acylamino, alkoxy, hydroxy, mercapto, phosphono).

[0232] (d). Alternatively the library may be assembled as BLFGs byreaction of the isocyanate (Z=O) or isothiocyanate (Z=S) LFGs withalcohols (March et al-, supra, pp. 891-892). R⁸ may be H, or a straightchain or branched alkyl groups of length 1 to 10, which may incorporatefrom 1 to 10 heteroatoms (N, O, P, S) within the chain. R⁸ may also beappended with up to five R⁹ groups (R⁹ is alkyl, aryl, heteroaryl,carboxy ester, carboxamide, amino, N-acylamino, alkoxy, hydroxy,mercapto, phosphono, sulphono). R⁸ may also be an aryl or heteroarylgroup that is optionally substituted (alkyl, aryl, heteroaryl, carboxyester, carboxamide, amino N-acylamino, alkoxy, hydroxy, mercapto,phosphono).

[0233] 9. The Halide or Related Sulfonate Group as the LinkageFunctional Group and the Corresponding Blocked Linkage Functional Groups

[0234] (a). The linkage functional group is the halide leaving group ora similarly reactive sulfonate (X═OSO;R where R is substituted orunsubstituted alkyl or aryl, e.g. CH, CF₃, phenyl-CH₃ and phenyl-NO₂)leaving group. These CTBFs may be available commercially, or may beprepared by a variety of known methods to those practiced in the art.

[0235] The halide and sulfonate (X═OSO₂R where R is substituted orunsubstituted alkyl or aryl, e.g. CH₃, CF₃, phenyl-CH, and phenyl-NO)CTBFs may be represented as follows.

[0236] (b). Alternatively the library may be assembled with amine BLFGs.

[0237] (i). The amine BLFGs may be prepared by substitution of theleaving group (X=halide or OSO₂R where R is substituted or unsubstitutedalkyl or aryl, e.g. CH, CF₃, phenyl-CH₃ and phenyl-NO₂) with amines Theleaving group may be attached to aromatic or heteroaromaticfunctionality, alkenyl functionality, or it could be attached toaliphatic functionality. A number of methods are known to thosepracticed in the art [e.g, March, et al. supra, pp. 656-657 and 411-413;Wagaw et al., J. Am. Chem. Soc., 119: 8451-8458 (1997) and referencestherein; and Ahman et al. Tetrahedron Letters, 38: 6363-6366 (1997)].When X is substituted upon aromatic and heteroaromatic functionality, anS_(N)Ar reaction may be performed. If the leaving group X is substitutedupon aromatic, heteroaromatic or alkenyl functionality, apalladium-mediated (or related transition metal mediated) amine couplingreaction may be performed. Where X is substituted upon alkylfunctionality, an S_(N)2 or S_(N)1 reaction may be performed. R⁸ or R⁹may be H, or a straight chain or branched alkyl group of length 1 to 10,which may incorporate from 1 to 10 heteroatoms (N, O, P, S) within thechain. R⁸ or R⁹ may also be appended with up to five R¹⁰ groups (R¹⁰ isalkyl, aryl, heteroaryl, carboxy ester, carboxamide, amino, N-acylamino,alkoxy, hydroxy, mercapto, phosphono, sulphono). R⁸ or R⁹ may also be anaryl or heteroaryl group that is optionally substituted (alkyl, aryl,heteroaryl, carboxy ester, carboxamide, amino, N-acylamino, alkoxy,hydroxy, mercapto, phosphono).

[0238] (ii). The amine BLFGs may also be prepared by a two step process.In the first step, substitution of the leaving group X with an amide,thioamide, urea, thiourea sulfonamide, carbamate, thiocarbamate (Z=SO₂,CO, CS, CO₂, COS, CSO, CONR¹¹, CSNR¹¹) is performed (see 0-0 below). Xmay be a halide or OSO₂R where R is substituted or unsubstituted alkylor aryl, e.g. CH₃, CF₃, phenyl-CH and phenyl-NO. The leaving group maybe attached to aromatic or heteroaromatic functionality, alkenylfunctionality, or it may be attached to aliphatic functionality. When Xis substituted upon aromatic or heteroaromatic functionality, an S_(N)Arreaction may be performed. When X is substituted upon aromatic,heteroaromatic, allylic or alkenyl a palladium-mediated (or relatedtranstion metal mediated) amine coupling reaction may be performed.Where X is substituted upon alkyl functionality, an S_(N)2 or S_(N)1reaction may be performed. Methods are known to those practiced in theart (March et al., supra, pp.425-427). In the second step, the acyl orsulfonyl functionality may be removed by methods known to thosepracticed in the art (such as, acidic or basic hydrolysis, or dissolvingmetal reactions for sulfonamides (Greene et al., supra, 349-357 and379-385). Milder conditions may be applied to more specialized groups,e.g., trifluoroacetamides may be cleaved by mild basic hydrolysis(Greene et al., supra, 353-354), and nitrosubstitutedbenzenesulfonamides may be cleaved by thiolate addition) to providesecondary amines (Fukuyama, et al., Tetrahedron Letters, 38: 5831-5834(1997) and references therein). Alternatively for some derivatives,reduction will provide tertiary amines (e.g., March, et al., supra, pp.1212-1213). R⁸ to R¹¹ may be H, or a straight chain or branched alkylgroup of length 1 to 10, which may incorporate from 1 to 10 heteroatoms(N, O, P, S) within the chain. R⁸ to R¹¹ may also be appended with up tofive R¹² groups (R¹² is alkyl, aryl, halide, heteroaryl, carboxy ester,carboxamide, amino, N-acylamino, alkoxy, hydroxy, mercapto, phosphono,sulphono). R⁸ to R¹¹ may also be an aryl or heteroaryl group that isoptionally substituted (alkyl, aryl, halide, heteroaryl, carboxy ester,carboxamide, amino, N-acylamino, alkoxy, hydroxy, mercapto, phosphono).

[0239] (c). Alternatively the library may be assembled with ether BLFGs.The ether BLFGs may be prepared by displacement of leaving group LFGswith an alcohol. X may be a halide or a sulfonate group (OSO₂R where Ris substituted or unsubstituted alkyl or aryl, e.g. CH₃, CF₃, phenyl-CH₃and phenyl-NO₂). The halide could be attached to aromatic orheteroaromatic functionality, alkenyl functionality, or it could beattached to an aliphatic functionality. When X is substituted uponaromatic or heteroaromatic functionality an S_(N)Ar reaction may beperformed. When X is substituted on aromatic, heteroaromatic, allylic,or alkenyl functionality a palladium-mediated, Cu mediated, or relatedtranstion metal mediated coupling reaction may be performed performed[(e.g., March, et al., supra, pp. 654-655) and Hartwig et al.Tetrahedron Lett., 38: pp. 8005-8008). Where X is substituted upon alkylfunctionality, an S_(N)2 or S_(N)1 reaction may be performed (e.g.,March, et al., supra, pp. 386-387 and 388-389). R⁸ may be H, or astraight chain or branched alkyl group of length 1 to 10, which mayincorporate from 1 to 10 heteroatoms (N, O, P, S) within the chain. R⁸may also be appended with up to five R⁹ groups (R⁹ is alkyl, aryl,heteroaryl, carboxy ester, carboxamide, amino, N-acylamino, alkoxy,hydroxy, mercapto, phosphono, sulphono). R⁸ may also be an aryl orheteroaryl group that is optionally substituted (alkyl, aryl,heteroaryl, carboxy ester, carboxamide, amino, N-acylamino, alkoxy,hydroxy, mercapto, phosphono).

[0240] (d). Alternatively the library may be assembled with thioetherBLFGs. The thioether BLFGs may be prepared by displacement of leavinggroup LFGs with an thiol. X may be a halide or a sulfonate group (OSO₂Rwhere R is substituted or unsubstituted alkyl or aryl, e.g. CH₃, CF₃,phenyl-CH, and phenyl-NO₂). The halide could be attached to aromatic orheteroaromatic functionality, alkenyl functionality, or it could beattached to an aliphatic functionality. When X is substituted uponaromatic or heteroaromatic functionality an S_(N)Ar reaction may beperformed. When X is substituted on aromatic, heteroaromatic, allylic,or alkenyl functionality a palladium-mediated, Cu mediated, or relatedtranstion metal mediated coupling reaction may be performed. Where X issubstituted upon alkyl functionality, an S_(N)2 or S_(N)1 reaction maybe performed. These methods are known to those practiced in the art[(March et al., supra, 407-409) and (Peach in Patai The Chemistry of theThiol Group, pt. 1, John Wiley & Sons, New York, 1974, pp. 721-735)]. R⁸may be H, or a straight chain or branched alkyl group of length 1 to 10,which may incorporate from 1 to 10 heteroatoms (N, O, P, S) within thechain. R⁸ may also be appended with up to five R⁹ groups (R⁹ is alkyl,aryl, heteroaryl, carboxy ester, carboxamide, amino, N-acylamino,alkoxy, hydroxy, mercapto, phosphono, sulphono). R⁸ may also be an arylor heteroaryl group that is optionally substituted (alkyl, aryl,heteroaryl, carboxy ester, carboxamide, amino, N-acylamino, alkoxy,hydroxy, mercapto, phosphono). The thioether may also be oxidized to themore water soluble sulfoxide or sulfone BLFGs (March et al., supra, pp.1201-1203).

[0241] (e). Alternatively the library may be assembled with ester BLFGs.The ester BLFGs may be prepared by displacement of leaving group LFGswith a carboxylate. X may be a halide or a sulfonate group (OSO₂R whereR is substituted or unsubstituted alkyl or aryl, e.g. CH₃, CF₃,phenyl-CH₃ and phenyl-NO₂). The halide could be attached to aromatic orheteroaromatic functionality, alkenyl functionality, or it could beattached to an aliphatic functionality. When X is substituted uponaromatic or heteroaromatic functionality an S_(N)Ar reaction may beperformed. When X is substituted on aromatic, heteroaromatic, allylic,or alkenyl functionality a palladium-mediated, Cu mediated, or relatedtranstion metal mediated coupling reaction may be performed. Where X issubstituted upon alkyl functionality, an S_(N)2′ or S_(N)1 reaction maybe performed. Methods for these transformations are known to thosepracticed in the art [e.g., (March, et al., supra, pp. 398-399) and(e.g., Greene, et al., supra, pp. 228-229)]. R⁸ may be H, or a straightchain or branched alkyl group of length 1 to 10, which may incorporatefrom 1 to 10 heteroatoms (N, O, P, S) within the chain. R⁸ may also beappended with up to five R⁹ groups (R⁹ is alkyl, aryl, heteroaryl,carboxy ester, carboxamide, amino, N-acylamino, alkoxy, hydroxy,mercapto, phosphono, sulphono). R⁸ may also be an aryl or heteroarylgroup that is optionally substituted (alkyl, aryl, heteroaryl, carboxyester, carboxamide, amino, N-acylamino, alkoxy, hydroxy, mercapto,phosphono).

[0242] (f). Alternatively the library may be assembled with thiolesterBLFGS. The ester BLFGs may be prepared by displacement of leaving groupLFGs with a thiolacid. X may be a halide or a sulfonate group (OSO₂Rwhere R is substituted or unsubstituted alkyl or aryl, e.g. CH₃, CF₃,phenyl-CH₃ and phenyl-NO₂). The halide could be attached to aromatic orheteroaromatic functionality, alkenyl functionality, or it could beattached to an aliphatic functionality. When X is substituted uponaromatic or heteroaromatic functionality an reaction may be performed.When X is substituted on aromatic, heteroaromatic, allylic, or alkenylfunctionality a palladium-mediated, Cu mediated, or related transtionmetal mediated coupling reaction may be performed. Where X issubstituted upon alkyl functionality, an S_(N)2 or S_(N)1 reaction maybe performed. R⁸ may be H, or a straight chain or branched alkyl groupof length 1 to 10, which may incorporate from 1 to 10 heteroatoms (N, O,P, S) within the chain. R⁸ may also be appended with up to five R⁹groups (R⁹ is alkyl, aryl, heteroaryl, carboxy ester, carboxamide,amino, N-acylamino, alkoxy, hydroxy, mercapto, phosphono, sulphono). R⁸may also be an aryl or heteroaryl group that is optionally substituted(alkyl, aryl, heteroaryl, carboxy ester, carboxamide, amino,N-acylamino, alkoxy, hydroxy, mercapto, phosphono).

[0243] (g). Alternatively the library may be assembled with urea orthiourea BLFGs. These BLFGs may be prepared by displacement of leavinggroup LFGs with a primary or secondary ureas (Z=O) or thioureas (Z=S). Xmay be a halide or a sulfonate group (OSO₂R where R is substituted orunsubstituted alkyl or aryl, e.g. CH., CF₃, phenyl-CF₃ and phenyl-NO₂).The halide could be attached to aromatic or heteroaromaticfunctionality, alkenyl functionality, or it could be attached to analiphatic functionality. When X is substituted upon aromatic orheteroaromatic functionality an S_(N)Ar reaction may be performed. WhenX is substituted on aromatic, heteroaromatic, allylic or alkenylfunctionality a palladium-mediated, Cu mediated, or related transtionmetal mediated coupling reaction may be performed. Where X issubstituted upon alkyl functionality, an S_(N)2 or S_(N)1 reaction maybe performed. R⁸ to R¹⁰ may be H, or a straight chain or branched alkylgroup of length 1 to 10, which may incorporate from 1 to 10 heteroatoms(N, O, P, S) within the chain. R⁸ to R¹⁰ may also be appended with up tofive R¹¹ groups (R¹¹ is alkyl, aryl, heteroaryl, carboxy ester,carboxamide, amino, N-acylamino, alkoxy, hydroxy, mercapto, phosphono,sulphono). R⁸ to R¹⁰ may also be an aryl or heteroaryl group that isoptionally substituted (alkyl, aryl, heteroaryl, carboxy ester,carboxamide, amino, N-acylamino, alkoxy, hydroxy, mercapto, phosphono).

[0244] (h). Alternatively the library may be assembled with sulfonamideBLFGs. The sulfonamide BLFGs may be prepared by displacement of leavinggroup LFGs with a sulfonamide. X may be a halide or a sulfonate group(OSO₂R where R is substituted or unsubstituted alkyl or aryl, e.g. CH₃,CF₃, phenyl-CH₃ and phenyl-NO₂). The halide could be attached toaromatic or heteroaromatic functionality, alkenyl functionality, or itcould be attached to an aliphatic functionality. When X is substitutedupon aromatic or heteroaromatic functionality an S_(N)Ar reaction may beperformed. When X is substituted on aromatic, heteroaromatic, allylic oralkenyl functionality a palladium-mediated, Cu mediated, or relatedtranstion metal mediated coupling reaction may be performed. Where X issubstituted upon alkyl functionality, an S_(N)2 or S_(N)1 reaction maybe performed. These methods are known to those practiced in the art[(March et al., supra, pp.425-427) and (Fukuyama et al., TetrahedronLetters, 38:5831-5834 (1997)]. R⁸ may be a straight chain or branchedalkyl group of length 1 to 10, which may incorporate from 1 to 10heteroatoms (N, O, P, S) within the chain. R⁸ may also be appended withup to five R⁹ groups (R⁹ is alkyl, aryl, heteroaryl, carboxy ester,carboxamide, amino, N-acylamino, alkoxy, hydroxy, mercapto, phosphono,sulphono). R⁸ may also be an aryl or heteroaryl group that is optionallysubstituted (alkyl, aryl, heteroaryl, carboxy ester, carboxamide, amino,N-acylamino, alkoxy, hydroxy, mercapto, phosphono).

[0245] (i). Alternatively the library may be assembled with carbamate,thiocarbamate, or related BLFGs. These BLFGs may be prepared bydisplacement of leaving group with the corresponding carbamate,thiocarbamate, or related BLFGs. X may be a halide or a sulfonate group(OSO₂R where R is substituted or unsubstituted alkyl or aryl, e.g. CH₃,CF₃, phenyl-CH, and phenyl-NO₂). The halide could be attached toaromatic or heteroaromatic functionality, alkenyl functionality, or itcould be attached to an aliphatic functionality. When X is substitutedupon aromatic or heteroaromatic functionality an S_(N)Ar reaction may beperformed. When X is substituted on aromatic, heteroaromatic, allylic oralkenyl functionality a palladium-mediated, Cu mediated, or relatedtranstion metal mediated coupling reaction may be performed. Where X issubstituted upon alkyl functionality, an S_(N)2 or S_(N)1 reaction maybe performed. R⁸ to R⁹ may be H, or a straight chain or branched alkylgroup of length 1 to 10, which may incorporate from 1 to 10 heteroatoms(N, O, P, S) within the chain. R⁸ to R⁹ may also be appended with up tofive R11 groups (R¹⁰ is alkyl, aryl, heteroaryl, carboxy ester,carboxamide, amino, N-acylamino, alkoxy, hydroxy, mercapto, phosphono,sulphono). R⁸ to R¹⁰ may also be an aryl or heteroaryl group thatis-optionally substituted (alkyl, aryl, heteroaryl, carboxy ester,carboxamide, amino, N-acylamino, alkoxy, hydroxy, mercapto, phosphono.

[0246] (j). Alternatively the library may be assembled by BLFGs wherethe leaving group is replaced with carbon-based functionality.

[0247] (i). X may be a halide or a sulfonate group (OSO₂R where R issubstituted or unsubstituted alkyl or aryl, e.g. CH₃, CF₃, phenyl-CH₃and phenyl-NO₂). The metal (M) may be any metal, but the prefered metalsare BL_(n), SnL_(n), ZnL_(n), ZrL_(n), CuL_(n), SiL_(n), Ti L_(n), AlL_(n), and L_(n) where L corresponds to metal ligands (e.g., halide,alkoxide, alkyl, aryl, alkenyl, heteroaryl, phosphine sulfide, amido)many of which are acceptable and are known to those practiced in theart. The halide could be attached to aromatic or heteroaromaticfunctionality, alkenyl functionality, or it could be attached to analiphatic functionality. Addition can occur directly, or can becatalyzed by transition metals. Numerous methods are known to thosepracticed in the art [e.g. (Hegedus, L. S. (1994) in Transition Metalsin the Synthesis of Complex Organic Molecules pp. 65-129, UniversityScience Books, Mill Valley), (Knochel et al. Chemical Reviews93:2117-2188 (1993)), (Miyaura et al. Chemical Reviews 95: 2457-2483(1995)), and (Farina et al.(Paquette, Series Editor in Chief), OrganicReactions, John Wiley & Sons, New York, 1997, vol. 50, pp. 1-653)]. R⁸may be H, or a straight chain or branched alkyl group of length 1 to 10,which may incorporate from 1 to 10 heteroatoms (N, O, P, S) within thechain. R⁸ may also be appended with up to five R⁹ groups (R⁹ is alkyl,aryl, heteroaryl, carboxy ester, carboxamide, amino, N-acylamino,alkoxy, hydroxy, mercapto, phosphono, sulphono). R⁸ may also be an arylor heteroaryl group that is optionally substituted (alkyl, aryl,heteroaryl, carboxy ester, carboxamide, amino, N-acylamino, alkoxy,hydroxy, mercapto, phosphono).

[0248] (ii). X may be a halide or a sulfonate group (OSO₂R where R issubstituted or unsubstituted alkyl or aryl, e.g. CH₃, CF₃, phenyl-CH₃and phenyl-NO₂). The BLFGs may be prepared in a two step process. In thefirst step, an organometallic reagent is prepared where the metal (M)may be any metal, but the prefered metals are BL_(n), SnL_(n), ZnL_(n),ZrL_(n), CuL_(n), SiL_(n), Ti L_(n), Al L_(n), and L_(n), where Lcorresponds to metal ligands (e.g., halide, alkoxide, alkyl, aryl,alkenyl, heteroaryl, phosphine sulfide, amido) many of which areacceptable and are known to those practiced in the art. In the secondstep, carbon-carbon bond formation is performed to generate the BLFGs.Numerous methods for this two step process are known to those practicedin the art [e.g. (Hegedus, L. S. (1994) in Transition Metals in theSynthesis of Complex Organic Molecules pp. 65-129, University ScienceBooks, Mill Valley), (Knochel et al. Chemical Reviews 93:2117-2188(1993)), (Miyaura et al. Chemical Reviews 95: 2457-2483 (1995)), and(Farina et al.(Paquette, Series Editor in Chief), Organic Reactions,John Wiley & Sons, New York, 1997, vol. 50, pp. 1-653)]. R⁸ may be H, ora straight chain or branched alkyl group of length 1 to 10, which mayincorporate from 1 to 10 heteroatoms (N, O, P, S) within the chain. R⁸may also be appended with up to five R⁹ groups (R⁹ is alkyl, aryl,heteroaryl, carboxy ester, carboxamide, amino, N-acylamino, alkoxy,hydroxy, mercapto, phosphono, sulphono). R⁸ may also be an aryl orheteroaryl group that is optionally substituted (alkyl, aryl,heteroaryl, carboxy ester, carboxamide, amino, N-acylamino, alkoxy,hydroxy, mercapto, phosphono).

[0249] (iii). X may be a halide or a sulfonate group (OSO₂R where R issubstituted or unsubstituted alkyl or aryl, e.g. CH₃, CF₃, phenyl-CH₃and phenyl-NO₂). The halide would preferably be attached to aromatic,heteroaromatic functionality, or alkenyl functionality. Apalladium-mediated Heck reaction or related transtion metal mediatedcoupling reaction would be performed [e.g., (de Meijere, A. et al.,Angew. Chem. Int. Ed. Engl., 33:2379-2411 (1994) and references therein)and Heck, Palladium Reagents in Organic Synthesis, Academic Press,London 1985)]. For the alkene, R⁸-R⁹ may be straight chain or branchedalkyl groups of length 1 to 10, which may incorporate from 1 to 10heteroatoms (N, O, P, S) within the chain. R⁸-R⁹ may also be appendedwith up to five R¹⁰ groups (R¹⁰ is alkyl, aryl, heteroaryl, carboxyester, carboxamide, amino, N-acylamino, alkoxy, hydroxy, mercapto,phosphono, sulphono). R⁸-R⁹ may also be aryl or heteroaryl groups thatare optionally substituted (alkyl, aryl, heteroaryl, carboxy ester,carboxamide, amino, N-acylamino, alkoxy, hydroxy, mercapto, phosphono).

[0250] (iv). X may be a halide or a sulfonate group (OSO₂R where R issubstituted or unsubstituted alkyl or aryl, e.g. CH₃, CF₃, phenyl-CH₃and phenyl-NO₂). The halide would preferably be attached to aromatic,heteroaromatic functionality, or alkenyl functionality. Acopper-mediated, palladium-mediated reaction, or related transtion metalmediated coupling reaction would be performed. Numerous methods areknown to those practiced in the art [e.g., (March et al., supra, p.481), (Sonagashira, K. in Comprehensive Organic Synthesis; Trost, B. M.,Fleming, I., Eds.; Pergamon Press: New York, 1991, vol. 3 pp. 521-549),and (Rossi et al., Org. Prep. Proced. Int. 27:129-160 (1995)]. For thealkyne, R⁸ may be H, straight chain or branched alkyl groups of length 1to 10, which may incorporate from 1 to 10 heteroatoms (N, O P, S) withinthe chain. R⁸ may also be appended with up to five R⁹ groups (R⁹is-alkyl, aryl, heteroaryl, carboxy ester, carboxamide, amino,N-acylamino, alkoxy, hydroxy, mercapto, phosphono, sulphono). R⁸ mayalso be a aryl or heteroaryl group that is optionally substituted(alkyl, aryl, heteroaryl, carboxy ester, carboxamide, amino,N-acylamino, alkoxy, hydroxy, mercapto, phosphono).

[0251] 10. The Alkenyl Group as the Linkage Functional Group and theCorresponding Blocked Linkage Functional Groups

[0252] (a). The linkage functional group is the alkenyl group. TheseCTBFs may be available commercially, or may be prepared by a variety ofknown methods to those practiced in the art.

[0253] CTBFs may be represented as follows, where as many as four CTBFfragments (A through D) may be present on the alkene functionality.

[0254] (b). Alternatively the library may be assembled with modifiedalkene BLFGs.

[0255] (i). The alkene BLFGs may be prepared by performing a transitionmetal-mediated (typically Pd) catalyzed Heck reaction or relatedreactions on trisubstituted, disubstituted or the vinyl functionalizedCTBFs [e.g., (de Meijere, A. et al., Angew. Chem. Int. Ed. Engl.,33:2379-2411 (1994) and references therein) and Heck, Palladium Reagentsin Organic Synthesis, Academic Press, London 1985)]. R⁸ may be astraight chain or branched alkyl group of length 1 to 10, which mayincorporate from 1 to 10 heteroatoms (N, O, P, S) within the chain. R⁸may also be appended with up to five R⁹ groups (R⁹ is alkyl, aryl,heteroaryl, carboxy ester, carboxamide, amino, N-acylamino, alkoxy,hydroxy, mercapto, phosphono, sulphono). R⁸ may also be a aryl orheteroaryl group that is optionally substituted (alkyl, aryl,heteroaryl, carboxy ester, carboxamide, amino, N-acylamino, alkoxy,hydroxy, mercapto, phosphono). X may be a halide or a sulfonate group(OSO₂R where R is substituted or unsubstituted alkyl or aryl, e.g. CH₃,CF₃, phenyl-CH₃ and phenyl-NO₂).

[0256] (ii). The alkene BLFGs may be prepared by performing a transitionmetal (typically Ru or Mo) catalyzed olefin metathesis reaction [e.g.,(Grubbs et al. Accounts of Chemical Research 28:446-452 (1995)) and(Schuster et al. Angewandte Chemie-International Edition in English36:2037-2056 (1997))]. R⁸-R¹⁴ may be straight chain or branched alkylgroups of length 1 to 10, which may incorporate from 1 to 10 heteroatoms(N, O, P, S) within the chain. R⁸-R¹⁴ may also be appended with up tofive R¹⁰ groups (R10 is alkyl, aryl, heteroaryl, carboxy ester,carboxamide, amino, N-acylamino, alkoxy, hydroxy, mercapto, phosphono,sulphono). R⁸-R¹⁴ may also be aryl heteroaryl groups that are optionallysubstituted (alkyl, aryl, heteroaryl, carboxy ester, carboxamide, amino,N-acylamino, alkoxy, hydroxy, mercapto, phosphono).

[0257] 11. Methods to Covalently Bond TBFs Using the LFG with aBifunctional Linker (BFL) to Produce Candidate Cross-linked TargetBinding Molecules (CXL-TBFs).

[0258] Upon identification of the TBFs, crosslinking is accomplishedwith a BFL. All of the chemistry described for preparation of the BLFGscould be employed in the crosslinking step. One method would be tocrosslink TBFs that have the same LFGs and employing the samecrosslinking chemistry. A second method would be to crosslink TBFs thathave the same LFGs, but employing different crosslinking chemistry. Athird method would be to crosslink TBFs that have different LFGs whichis most but not all cases would require different corsslinkingchemistry. Each of these strategies use known methods to those practicedin the art. Examples of the three methods are provided below.

[0259] Method 1. For example, two TBFs with aldehyde LFGs could becross-linked employing a BFL that incorporates two O-substitutedhydroxylamines.

[0260] Method 2. For example, two TBFs with aldehyde LFGs could becross-linked employing a BFL that incorporates one O-substitutedhydroxylamine and one acyl hydrazide.

[0261] Method 3. For example, one TBF with an aldehyde LFG and one TBFwith an amine LFG could be cross-linked employing a BFL thatincorporates one O-substituted hydroxylamine and one carboxylic acid.

[0262] A hypothetical example for the preparation of heterolinkers wouldbe to employ amino acids as heterolinkers. As shown below, the aminoacid heterolinker serves to link TBFs with carboxylic acid LFGs to TBFswith amine LFGs. The amine LFG could be a primary amine or a secondaryamine (not shown). Many methods that are known to those practiced in theart could be used to prepare the CXL-TBFs using amino acidheterolinkers. One of the methods is described below.

[0263] For this sample method, the amino acid BFL is protected as theN-tertbutoxycarbonyl (Boc) derivative. Many N-Boc amino acids arecommercially available, e.g., Novabiochem (San Diego, Calif.) andNeosystems (Strosbourg, France). N-Boc protected amino acids can also beprepared by known methods to those practiced in the art (Bodansky etal., The Practice of Peptide Synthesis, Springer-Verlag, Berlin, 1984,18-20).

[0264] The sample method is illustrated in Scheme 1. Sample experimentalprocedures are provided below.

Step 1 (Desai, et al., Tetrahedron Letters, 34, 7685-7688, (1993))

[0265] To a suspension of polymer-bound carbodimide (1.5 mmol) inchloroform (10 mL) is added the N-Boc protected amino acid 2 (0.55 mmol)and the amine-substituted TBF 1 (0.50 mmol). After the reaction mixtureis shaken overnight at room temperature, the mixture is filtered. Theresin is washed with chloroform (3×7.5 mL) and the combined filtrate isevaporated in vacuo to yield 3.

Step 2

[0266] To compound 3 (0.5 mmol) is added 5 mL of a solution of 4.0 Mhydrochloric acid in dioxane (Aldrich, Milwaukee, Wis.). The solution isstirred for one hour at room temperature and then evaporated to removethe solvent and excess hydrochloric acid. The product is diluted with 5mL of methanol and concentration is repeated to provide 4.

Step 3 (Desai, et al., Tetrahedron Letters, 34, 7685-7688, (1993))

[0267] To a suspension of polymer-bound carbodiimide (1.5 mmol) inchloroform (10 mL) is added intermediate 4 (0.50 mmol) and thecarboxylic acid-substituted TBF 5 (0.55 mmol). After the reactionmixture is shaken overnight at room temperature the mixture is filtered.The resin is washed with chloroform (3×7.5 mL) and the combined filtrateis evaporated in vacuo to yield product 6, which is the desired CXL-TBF.

Experimental

[0268] Unless otherwise noted, materials were obtained from commercialsuppliers and used without further purifications. Aldehydes werepurchased from Aldrich Chemical Company, Inc. (Milwaukee, Wis.).Anhydrous dimethylsulfoxide (DMSO) and acetic acid were purchased fromFischer (Pittsburg, Pa.). Soluble CD4 (sCD4) was purchased from IntracelCorporation (Issaquah, Wash.), gp120 and anti-gp120 antibody werepurchased from DuPont (Wilmington, Del.) and o-phenylenediamineperoxidase substrate tablet sets were purchased from Sigma Chemical Co.(St. Louis, Mo.). Reactions were carried out in commercially availableBeckman 2 ml deep-well microtiter plates.

EXAMPLE I Pharmacophore Recombination for the Identification ofCompounds Capable of Inhibiting the Interaction Between zp120 and CD4

[0269] To demonstrate the principle of pharmacophore recombination, weestablished a biochemical screen for the inhibition of gp120-CD4binding. This assay measures the ability of small molecules to inhibitthe binding of gp120 to sCD4 that is immobilized on a microtiter plate.Binding of sCD4 was quantified with an antigp120 antibody conjugated tohorseradish peroxidase.

[0270] General Procedure for the Synthesis of an Oxime Compound Library

[0271] For several reasons, we chose to initially employ O-methyloximes, rather than aldehydes, for the initial compound building blocklibrary. First, O-methyl oximes best model the pharmacophore units inthe final oxime coupled dimers. Second, O-methyl oximes are more solublein aqueous solution than their more hydrophobic aldehyde precursors.Also, the oxime functionality is clearly not inherently toxic and doesnot interfere with good pharmacokinetics or cell permeability sinceoximes are present in many drugs. Finally, the O-methyl oximes areeasily prepared in a single step condensation of aldehydes with O-methylhydroxylamine, without requiring purification of the resultant product.The chemical condensation of an aldehyde with O-methyl hydroxylamine toprovide an oxime compound is shown in FIG. 1.

[0272] In the first step of the method, the initial oxime library wassynthesized by separately condensing O-methyl hydroxylamine with 252different aldehydes in a DMSO solution. The oxime library was preparedin a spatially separate fashion in a microtiter plate format such thateach well contained a single oxime compound. More specifically, in eachwell of a microtiter plate, a DMSO solution of an unique aldehyde (0.188ml, 0.15 M, 0.028 mmol) was added. To this solution, a DMSO solution ofO-methyl hydroxylamine (0.083 ml, 0.5 M, 0.042 mmol) was then addedfollowed by addition of a DMSO solution of acetic acid (0.023 ml, 0.5 M,0.011 mmol). The plates were allowed to sit at room temperatureovernight during which time condensation occurred, thereby providing the252 member library of oxime compounds.

[0273] Assay to Determine which Oxime Compounds are Capable ofInhibiting the Interaction Between gp120 and CD4

[0274] The 252 member oxime compound library prepared as described abovewas then screened for the presence of compounds capable of inhibitingthe interaction between gp120 and CD4 in a standard ELISA assay. For thegp120-CD4 ELISA assay, an Immulon-2 microtiter plate was incubatedovernight at 4° C. with 70 ng of sCD4 in 100 μl of carbonate buffer. Thesolution was removed from the plate and washed three times withphosphate buffered saline (PBS) at pH 7.4. The plate was blocked with150 μl PBS-Tween-BSA (0.5% BSA, 0.05% Tween-20) for 1 h at roomtemperature and then washed again. gp120 (1 ng) in 50 μl of PBS and 50μl of test organic oxime compound (3 nM), 40 oil PBS, 10 μl were addedand incubated for 1 h at room temperature. The plate was then washed and100 μl of anti-gp120 conjugated horseradish peroxidase was added andincubated for 1 h at room temperature. The bound gp120 was thenquantitated with o-phenylenediamine as a substrate.

[0275] The results of these assays demonstrated that 30 of the 252 oximecompounds were capable of inhibiting the interaction between gp120 andCD4, wherein the approximate EC₅₀ values ranged from about 20 μM to 500μM. The structurally related aldehyde analogs of 30 of these oximesshowed diverse structural motifs including chromones, phenols and furans(see FIG. 2).

[0276] Cross-Linking of the Top 30 Structurally Related Aldehyde Analogsto Produce a Library of Candidate Compounds and Screening of ThoseCandidate Compounds for the Ability to Inhibit the Interaction Betweengp120 and CD4

[0277] Each of the 30 structurally related aldehydes analogscorresponding to the 30 oxime compounds identified above as beingcapable of inhibiting the interaction between gp120 and CD4 wereindividually coupled to each of the other 29 aldehydes with a variety oflinkers to produce a library of candidate compounds for binding to thetarget molecule. Each of the 30 individual aldehydes was linked withanother aldehyde through an O,O′-diamino-alkanediol linker to obtain thelibrary of cross-linked candidate compounds. Each aldehyde combinationwas kept spatially separate, but an equimolar mixture of five differentO,O′-diamino-alkanediol linkers were used in each coupling reaction toprovide a 450-member library of cross-linked candidate compounds. Thechemistry used for preparation of the compounds is shown in FIG. 3showing the synthesis of both homodimers and heterodimers.

[0278] The five linkers employed each consisted of two hydroxylaminegroups tethered to an aliphatic chain having either two, three, four,five or six methylene units. This allowed us to evaluate any distancedependency the two pharmacophores may have in the binding site. Linkerswere prepared as follows. To a round-bottomed flask was added alkyldibromide (20.2 mmol), N-hydroxyphthalimide (36.8 mmol, ˜1.8 equiv) anddimethylformamide (90 ml). The flask was cooled to 0° C. and1,8diazabicyclo[5.4.0]undec-7-ene (40.5 mmol) was added dropwise withstirring. The reaction was allowed to warm to room temperature and wasthen stirred overnight. The reaction mixture was then poured into 1M HCl(500 ml). The resulting white solid precipitate was washed with water(3×50 ml) and methanol (3×50 ml) and sent onto the next step withoutfurther purification.

[0279] 13.9 mmol of the crude bis-N-alkoxyphthalimide was then added toa round-bottomed flask in combination with dimethoxyethylene glycol (150ml). To another flask was added hydrazine monohydrate (41.8 mmol, 3equiv.) and dimethoxyethylene glycol (100 ml). The suspension ofbis-N-alkoxyphthalimide was added slowly with stirring to the hydrazinesolution. The flask was refluxed for 3 h, allowed to cool to roomtemperature and the resulting precipitate was filtered away. Theremaining supernatant solution was concentrated and the resultingyellowish-oil was purified by Kugelrohr distillation (0.01 mm Hg, 60-70°C.) and column chromatography (89:9:2 CHCl₃/MeOH/NH₄OH).

[0280] For O,O′-diamino-1,4-butanediol), the general synthesis proceduredescribed above was followed. IR (film from CHCl₂): 3412.8, 3310.0,2942.7, 2866.3 cm¹. ¹H NMR (400 MHz, CDCl₃): δ 5.31 (br s, 4H), 3.65 (m,4H), 1.57 (m, 4H). ¹³C NMR (400 MHz, CDCl₃): δ 75.5, 24.8. Anal. Calcdfor C₄H₁₂O₂N₂: C, 39.99; H, 10.07; N, 23.32. Found: C, 40.17; H, 9.90;N, 23.12.

[0281] Once the five linkers were obtained, the 450 linked aldehydecombinations were prepared as follows. In each well, a DMSO solution ofeach of two different aldehydes (0.045 ml, 0.15 M, 0.007 mmol each) wasadded. To this solution, a DMSO solution of an equimolar mixture of thefive linkers (0.025 ml, 0.3 M of each linker, 0.007 mmol) was addedfollowed by a DMSO solution of acetic acid (0.005 ml, 0.5 M, 0.003mmol). The plates were allowed to sit at room temperature overnight toallow for potential ligand formation.

[0282] Each of the 450 members of the potential ligand library was thentested at a concentration of 100 μM each (i.e., a concentration that is10-fold more dilute than the concentration employed in the initial oximemonomer screen) for the ability to inhibit the interaction between gp120and CD4 using the ELISA assay described above. The results from theseassays demonstrated that more than 300 of the 450 members of thepotential ligand library showed greater than 50% inhibition activity atthe concentration employed. When the 450 members of the potential ligandlibrary were tested at a concentration of 1 μM each for the ability toinhibit the interaction between gp120 and CD4, 17 of the ligands showedgreater than 50% inhibitory activity at that concentration. The chemicalstructures of 12 of the 17 most active aldehyde precursors are shown inFIG. 4.

[0283] Evaluation of Linker Length Dependence on Activity

[0284] The 17 linked aldehyde combinations with the greatest activitywere then resynthesized as described above with a unique linker per well(85 separate wells) so as to evaluate any linker-length dependency onbinding. Screening each of the 85 ligand compounds at 1 μM each showedthat ligands that incorporated pharmacophore 1 (see FIG. 5) were morepotent than the other ligands and that there indeed was a dependenceupon linker length. Specifically, ligands with linkers having either 4or 5 methylene units were much more active than ligands with linkershaving 6 methylene units, whereas ligands with linkers having either 2or 3 methylene units were only slightly more active than ligands withlinkers having either 4 or 5 methylene units.

[0285] Biological and Analytical Characterization of RepresentativeLigands

[0286] One of the ligands that exhibited strong activity for inhibitingthe interaction between gp120 and CD4 (as shown in FIG. 6) wasresynthesized with each of the five different linkers on a large scaleand was then purified by column chromatography. Column chromatographypurification enabled isolation of the heterodimer from the homodimer.

[0287] Large scale synthesis of organic oxime compounds was performed asfollows. To a flame-dried round-bottomed flask was added aldehyde (0.82mmol) and DMSO (8 ml). A 0.9 M O-methyl hydroxylamine (1.4 ml) was thenadded and the reaction mixture was allowed to stir at room temperatureovernight. The reaction was poured into methylene chloride (50 ml),washed with H₃O (3×20 ml), dried and concentrated. Silica gelchromatography provided the pure organic oxime compounds.

[0288] The oxime compounds made by this method were characterized asfollows.

[0289] (1) O-Methyl Oxime of 6-Nitropiperonal

[0290] Reaction of 6-nitropiperonal with O-methyl hydroxylamine providedpredominantly one oxime isomer which was purified by silica gelchromatography (10:90, EtOAc/hexanes). ¹H NMR (400 MHz, CDCl₃): δ 8.61(s, 1H), 7.53 (s, 1H), 7.37 (s, 1H), 6.15 (s, 2H), 4.00 (s, 3H). Anal.Calcd for C₉H₈O₅N₂: C, 48.22; H, 3.60; N, 12.50. Found: C, 48:40; H,3.75; N, 12.56.

[0291] (2) O-Methyl Oxime of 6,8-dichloro-3-formylchromone

[0292] Reaction of 6,8-dichloro-3-formylchromone provided 1:1 cis/transisomers which were isolated by silica gel chromatography (20:80,CH₂Clexanes).

[0293] Isomer 1: ¹H NMR (400 MHz, CDCl₃): δ 8.48 (s, 1H), 8.24 (s, 1H),8.12 (d, 1H, J=2.5), 7.75 (d, 1H, J=2.5), 3.97 (s, 3H). Anal. Calcd forC₁₁O₃NCl₂: C, 48.56; H, 2.59; N, 5.15. Found: C, 48.44; H, 2.47; N,5.03.

[0294] Isomer 2: ¹H NMR (400 MHz, CDCl₃): δ 9.45 (s, 1H), 8.12 (d, 1H,J=2.5), 7.75 (d, 2H, J=2.5), 4.07 (3H). Anal. Calcd for C₁₁O₃NCl₂: C,48.56; H, 2.59; N, 5.15. Found: C, 48.45; H, 2.49; N, 5.11.

[0295] Large scale synthesis of oxime ligands was performed as follows.To a flame-dried round-bottom flask was added 10 ml of DMSO and 1.03mmol of each of the two aldehydes to be incorporated into the ligand.After all solids were dissolved, a solution of the appropriate linker(1.24 mmol) in 1 ml of DMSO was added dropwise, followed by the additionof acetic acid (0.72 mmol). The reaction mixture was allowed to stir atroom temperature overnight. The reaction was then poured into methylenechloride (50 ml), washed with H₂O (3×20 ml), dried and concentrated.Silica gel chromatography provided the isolated homo/heterodimers.Cis/trans isomers, when present, were not separated and were purified asmixtures of isomers. The oxime dimers made by this method werecharacterized below.

[0296] 1) Oxime Heterodimer of 6,8-dichloro-3-formylchromone and6-nitropiperonal, Linker Containing 4 Methylene Units

[0297] The heterodimer was separated from the homodimers by silica gelchromatography (20:80, EtOAc/hexanes). The heterodimer was isolated andcharacterized as a 1:1 mixture of cis/trans isomers. Anal. Calcd forC₂₂H₁₇O₈N₃Cl₂: C, 50.59; H, 3.28; N, 8.05. Found: C, 50.70; H, 3.40; N,7.89.

[0298] Isomer 1: ¹H NMR (400 MHz, CDCl₃): δ 9.42 (s, 1H), 8.55 (s, 1H),8.00 (s, 1H), 7.67 (m, 2H), 7.39 (s, 1H), 7.27 (s, 1H), 6.09 (s, 2H),4.22 (m, 4H), 1.84 (m, 4H).

[0299] Isomer 2: ¹H NMR (400 MHz, CDCl₃): δ 8.54 (s, 1H), 8.41 (s, 1H),8.16 (s, 1H), 7.99 (s, 1H), 7.67 (s, 1H), 7.40 (s, 1H), 7.24 (s, 1H),6.08 (s, 2H), 4.22 (m, 4H), 1.83 (m, 4H).

[0300] (2) Oxime Heterodimer of 6.8-dichloro-3-formylchromone and6-nitropiperonal, Linker Containing 5 Methylene Units

[0301] The heterodimer was separated from the homodimers by silica gelchromatography (20:80, EtOAc/hexanes). The heterodimer was isolated andcharacterized as a 1.5:1 mixture of cis/trans isomers. Anal. Calcd forC₂₃H₁₉O₈N₃Cl₂: C, 51.51; H, 3.57; N, 7.83. Found: C, 51.68; H, 3.70; N,7.66.

[0302] Isomer 1: ¹H NMR (400 MHz, CDCl₃): δ 8.62 (s, 1H), 8.47 (s, 1H),8.24 (s, 1H), 8.10 (s, 1H), 7.74 (s, 1H), 7.73 (s, 1H), 7.37 (s, 1H),6.14 (s, 2H), 4.20 (m, 4H), 1.8 (m, 4H), 1.53 (m, 2H).

[0303] Isomer 2: ¹H NMR (400 MHz, CDCl3): δ 9.47 (s, 1H), 8.61 (s, 1H),8.11 (s, 1H), 7.75 (s, 2H), 7.48 (s, 1H), 7.33 (s, 1H), 6.14 (s, 2H),4.30 (t, 2H, J=6.6), 4.20 (m, 2H), 1.80 (m, 4H), 1.53 (m, 2H).

[0304] These purified heterodimers and homodimers were then tested asdescribed above for the ability to inhibit the interaction between gp120and CD4. The results of these assays demonstrated that heterodimersshown in FIG. 6 having a linker containing from 2 to 5 methylene unitsexhibited EC₅₀'s ranging from 0.6 to 1.5 μM and showing 10- to 20-foldenhancement in inhibitory activity over the compound shown in FIG. 5(EC₅₀ in the range of about 10-15 μM). The other compound that wasincorporated into the heterodimer had an EC₅₀ of greater than 50 μM.

[0305] The heterodimers shown in FIG. 6 having linkers containing from 2to 5 methylene units are of comparable potency to the most potentcompounds that have been identified to date that block the CD4/gp120interaction (Tanaka et al., J. Antibiotics 50:58 (1997), Sun et al., J.Antibiotics 49:689 (1997), Jarvest et al., Bio. Med. Chem. Lett. 3:2851(1993) and Chen et al., Proc. Natl. Acad. Sci. USA 89:5872 (1992)). Inaddition, these ligand heterodimers are considerably less complex thanpreviously identified compounds with comparable activity. Furtheroptimization of the optimal building block and linker combinations couldpresumably be accomplished by evaluating a larger range of linkers withenhanced rigidity or by incorporating analogs of the optimal aldehydeprecursors.

EXAMPLE 2 Pharmacophore Recombination Using N,N-Dimethylamines and OtherDiamine Linkers

[0306] In addition to the use of aldehydes and oximes for thepharmacophore recombination method as described above, additionalchemistries also find use. In this example, the organic compoundbuilding blocks are N,N-dimethylamine compounds that are prepared byreductive animation of starting aldehydes and dimethylamine usingsupport-bound triacetoxyborohydride (Kaldor et al., Tetrahedron Lett.37:7193-7196 (1996)). The chemistry of these reactions is shown in FIG.7. Removal of the support-bound reducing agent by filtration followed byconcentration to remove the volatile, excess dimethylamine then providesthe pure N,N-dimethylamine monomer building blocks. Alternatively, theN,N-dimethylamine building blocks may be obtained by reduction using asodium borohydride-based reducing agent in solution. The resulting amineproduct is then isolated from the excess reducing agent or aldehyde bypassing down an acidic ion exchange column. The amine product is thenobtained by elution from the ion exchange column with a volatile aminesuch as ammonia followed by concentration.

[0307] Linkage of the N,N-dimethylamine building blocks can beaccomplished through the use of diamine linkers of which many arecommercially available and many more can be readily prepared using wellknown methodology. The commercial availability of the diamine linkersallows rapid optimization of linker length, rigidity and orientation. Anexemplary synthesis sequence is shown in FIG. 8. Specifically,support-bound chloride (3) (or other support-bound halide) is treatedwith excess of a diamine to provide an amine-derivatized support (4).Acylation of the amine functionality then provides support-boundformamide or carbamate (5). Reduction then provides support-boundsecondary amine (6). Reductive amination then introduces one of thepharmacophore elements (7). Acid treatment then releases a secondaryamine from the support, which can then be treated with the secondpharmacophore monomer and sodium triacetoxyborohydride to provide thedesired pharmacophore heterodimer (either support-bound reagent oralternative scavenging methods may be employed). Initial attachment ofthe diamine to the support can be accomplished using other support-boundalkyl halides or could be accomplished by reductive amination of asupport-bound aldehyde or ketone. Fewer linkers are available thatcontain two secondary amine groups, but these can also be incorporated.in this case, the acylation step (4 to 5 in FIG. 8) and the subsequentreduction step (5 to 6 in FIG. 8) would be eliminated.

[0308] The foregoing description details specific methods which can beemployed to practice the present invention. Having detailed suchspecific methods, those skilled in the art will well enough know how todevise alternative reliable methods at arriving at the same informationin using the fruits of the present invention. Thus, however, detailedthe foregoing may appear in text, it should not be construed as limitingthe overall scope thereof; rather, the ambit of the present invention isto be determined only by the lawful construction of the appended claims.All documents cited herein are expressly incorporated by reference.

What is claimed:
 1. A method for screening a library of small organicmolecules for one or more candidate target binding fragments (CTBF's)that bind to a target biological molecule (TBM), each CTBF having alinkable functional group (LFG) or blocked form thereof (BLFG), whereinthe LFG or BLFG contains a disulfide linking group (LG), the methodcomprising: (a) contacting the TBM with individual members of a libraryof the CTBF; (b) detecting or determining which CTBF's bind to the TBM;and (c) selecting CTBF's that bind to the TBM.
 2. The method of claim 1,wherein (b) comprises quantifying the binding association of the CTBF'swith the TBM.
 3. The method of claim 2, wherein a quantitativespectroscopic property is used to determine the binding association ofthe CTBF's with the TBM.
 4. The method of claim 1, wherein (b) isaccomplished by an in vitro biological assay.
 5. The method of claim 4,wherein (b) comprises an ELISA assay.
 6. The method of claim 1, whereinX-ray crystallography is used to determine the binding association ofthe CTBF's with the TBM.
 7. The method of claim 1, further comprisingsubjecting the bound CTBF's and TBM to X-ray crystallography.
 8. Themethod of claim 1, wherein each CTBF further contains a second LGselected from the group consisting of amide, secondary amine, disulfide,sulfonamide, ureido, thiourea, carbamate and sulfonamide.
 9. The methodof claim 1, further comprising linking at least two of the selectedCTBF's or analogs thereof.
 10. The method of claim 1, further comprisingconverting the selected CTBF's to structurally related analogs thereof.11. The method of claim 1, further comprising linking the selectedCTBF's to a second compound.
 12. The method according to claim 1,wherein the TBM is a protein.
 13. The method according to claim 12,wherein the protein is a hormone, cytokine, chemokine or receptor. 14.The method according to claim 1, wherein the TBM is an enzyme.
 15. Themethod according to claim 14, wherein the enzyme is a protease,phosphatase (dephosphorylase) or kinase.
 16. The method according toclaim 1, wherein the library of CTBF's comprises small organic moleculeswith molecular weights of less than about 1000 Daltons.
 17. The methodaccording to claim 16, wherein the library of CTBF's comprises smallorganic molecules with molecular weights of less than about 500 Daltons.18. The method of claim 17, wherein the library of CTBF's for binding toa TBM comprises at least about 100 different CTBF's.